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Background: Implementation of respiratory virus prevention measures requires detailed understanding of regional epidemiology; however, data from many tropical countries are sparse. We describe etiologies of ambulatory pediatric acute respiratory tract infections (ARTI) in Ecuador immediately preceding the onset of the SARS-CoV-2 pandemic., Methods: Children < 5 years presenting to a designated study site with an ARTI were eligible. Informed consent was obtained. Demographic and clinical data were recorded. A nasopharyngeal swab was collected, processed, and analyzed using multiplex polymerase chain reaction (PCR) for common respiratory pathogens. Rhinovirus/enterovirus positive samples were further characterized by genomic sequencing., Results: A total of 820 subjects were enrolled in the study between July 2018 and March 2020. A total of 655 (80%) samples identified at least one pathogen. Rhinoviruses (44%) were most common, followed by enteroviruses (17%), parainfluenza viruses (17%), respiratory syncytial virus (RSV) (15%), and influenza viruses (13%). Enterovirus D68 was the most common enterovirus detected and was among the leading causes of bronchiolitis. Seasonal RSV and influenza virus activity were different along the coast compared with the highlands., Conclusions: Ongoing regional surveillance studies are necessary to optimize available and emerging pathogen-specific preventative measures., (© 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Objective: We introduced a multi-component cancer prevention awareness program to primary care practices across New York State to evaluate its impact on adolescent human papillomavirus (HPV) vaccination rates., Methods: Eight pediatric and three family medicine practices were recruited to participate in this program. On-site training sessions were provided for all practice providers and staff to discuss the importance of HPV vaccine and cancer prevention and teach strategies for delivering a strong vaccine recommendation. Each practice received a study-specific booklet that included HPV vaccine information and other commonly provided cancer prevention guidance. These booklets were distributed to all adolescents and their parents during well visits over a one-year period. Practice specific and county-wide HPV vaccination rates were assessed before and 12 months after the program training session., Results: One year after program initiation, aggregate data show statistically higher vaccine series initiation rates among 11-12 and 13-18-year-olds and higher vaccine series completion rates among 13-18-year-olds. The greatest and most consistent improvements were seen in vaccine initiation rates for the 11-12-year-old cohort. Disparities in vaccine uptake were observed by gender and medical specialty., Conclusion: Cancer prevention education targeting providers, office staff, patients, and parents, improved adolescent HPV vaccine series initiation rates.

Background: Human coronaviruses (HCoVs) cause respiratory tract infections during childhood manifesting as common colds, bronchiolitis, croup and pneumonia. In temperate geographies, HCoV activity peaks between December and March. The epidemiology and manifestations of HCoV infections have not been previously reported from Ecuador., Methods: Children <5 years who presented with ≥2 symptoms consistent with an acute respiratory tract infection were eligible for enrollment. After obtaining informed consent, demographic data and details regarding the acute illness were recorded. Secretions collected with a nasopharyngeal swab underwent diagnostic testing using multiplex polymerase chain reaction., Results: A total of 850 subjects were enrolled. A total of 677 (80%) tested positive for at least 1 pathogen, including 49 (7.2%) who tested positive for ≥1 HCoV type. HCoV-NL63 was the most frequent type detected (39%), followed by HCoV-OC43 (27%), 229E (22%) and HKU1 (12%). Nearly all subjects who tested positive for HCoV had nasal congestion or secretions (47/49; 96%). The most frequent syndromic diagnosis was common cold (41%), followed by bronchiolitis (27%). We found no association between the infecting HCoV type and subject's syndromic diagnosis (P > 0.05) or anatomic location of infection (upper vs. lower respiratory tract; P > 0.05). The 2018-2019 peak HCoV activity occurred from October to November; the 2019-2020 peak occurred from January to February., Conclusions: HCoVs were detected in ~7% of outpatient Ecuadorean children <5 years of age with symptoms of acute respiratory tract infection. The most frequently detected HCoV types, and the period of peak HCoV activity differed for the 2018-2019 and 2019-2020 seasons.

Background: Politics play a role in the dissemination of public health information, including immunization-related issues. We aim to describe relationships between HPV vaccination rates and state voting patterns during the 2016 US presidential election., Methods: We classified each of the 50 states as either "Red" or "Blue," based on whether a higher proportion of the state's casted votes were for the Republican or Democratic nominee during the 2016 US presidential election. State-specific HPV, Tdap, and meningococcal vaccination rates were obtained from the 2016-National Immunization Survey-Teen. State socio-demographic factors and HPV vaccine legislation were obtained from the US Census Bureau and National Conference of State Legislatures. Vaccination rates and socio-demographic variables were compared using independent t-tests. Multiple linear regression compared vaccination rates between "Red" and "Blue" states, adjusting for percentage of both uninsured children and educational attainment., Results: Compared to "Blue" states, "Red" states had significantly lower unadjusted HPV vaccine series initiation (56% vs 66%, p < 0.05) and completion (39% vs 50%, p < 0.05) rates; yet had similar rates of Tdap (88% vs 89%, p > 0.05) and meningococcal (79% vs 83%, p > 0.05) vaccinations. After adjusting for potential confounders, the regression-adjusted mean rate for HPV vaccine initiation and completion remained significantly lower for "Red" states compared to "Blue" states (57% vs 65%, p < 0.05, and 41% vs 48%, p < 0.05, respectively)., Conclusion: HPV vaccination rates are associated with statewide-level voting patterns. Future interventions aimed at improving HPV vaccination rates should consider engaging local and national elected leaders to be proactive in disseminating accurate and authoritative immunization information., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To develop a program to educate providers, office staff, patients, and parents on life-long cancer prevention strategies, including the use of human papillomavirus (HPV) vaccine to improve adolescent HPV vaccination rates., Study Design: A 2-phase program was implemented at 6 pediatric practices across upstate New York. Phase 1 included provider and staff education regarding practice-specific vaccination challenges and discussion of the contents of a study-specific cancer-prevention booklet, which included HPV vaccine information. Throughout phase 2, the booklets were distributed to all adolescents and their parents during office visits over a 12-month period. Practice-specific, countywide, and statewide HPV vaccination rates were assessed before phase 1, and 6 and 12 months after the launch of phase 2., Results: One year after implementing phase 2 in 6 practices, adolescent HPV vaccine series initiation increased by at least 10% in 3 practices, and at least 5% in 5 practices. Similarly, adolescent vaccine series completion rates increased by more than 10% in 3 practices. The percent change in vaccine series completion rates across all study sites postintervention ranged from 12% to 20% for 11- to 12-year-olds, and from 7% to 23% for 13- to 18-year-olds., Conclusions: Cancer prevention education targeting providers, office staff, patients, and parents was modestly effective for improving adolescent HPV vaccination rates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

HPV vaccine uptake is low, nationwide. Quality improvement (QI) principles have the potential to change practice; however, not all providers are confident with QI skills. We developed an educational program designed to enhance QI skills and improve HPV vaccination rates. Five pediatric practices participated in the pilot initiative. Training consisted of presentations regarding QI methods, data tracking and analysis, and system changes to reduce missed opportunities. Monthly for 6 months, participants performed chart audits, captured data, printed run charts, and developed, implemented, and tracked interventions. Outcome measures included rates of HPV vaccine completion and missed opportunities. A second phase included eight different pediatric practices who received similar training. Outcome measures included rates of HPV vaccine initiation and completion. Over the 6 months, mean HPV vaccine completion rates increased (45% to 65%) and missed opportunities for HPV vaccination decreased (45% to 19%) in the pilot program. When the program was replicated in phase 2, an increase was seen in both HPV vaccine initiation (46% to 61%) and completion (62% to 94%) rates. Combining QI education with workflow-focused strategies was associated with a reduction in missed opportunities and a substantial increase in HPV vaccine completion rates.

Respiratory syncytial virus (RSV) infection in infants is a major cause of morbidity and mortality worldwide. Despite intense research efforts, safe and effective vaccines have remained elusive. Risk factors for the development of severe disease are well known, and those infants at highest risk are identified to receive RSV prophylaxis in the form of anti-RSV monoclonal antibody. Still, many other infant groups remain at risk and could benefit from an effective RSV prevention program. An explosion of clinic research activity is bringing unprecedented progress. Several of the most promising approaches currently being evaluated in clinical trials are reviewed. [Pediatr Ann. 2018;47(9):e371-e376.]., (Copyright 2018, SLACK Incorporated.)

Objective: To describe immunization attitudes and practices among family medicine providers across New York State., Methods: In this cross-sectional survey study, family medicine providers across New York State completed a questionnaire to assess vaccine beliefs and barriers and immunization practices., Statistical Analysis: Descriptive statistical methods were used to define provider characteristics, knowledge and vaccine practices., Results: Completed questionnaires from 226 family medicine providers were included for analysis. As a group, 207/218 (95%) of providers who answered the question state they always recommend standard pediatric vaccines. Of the 209 providers who answered both questions, 47 (22%) state they always recommend standard pediatric vaccines but do not always recommend HPV vaccine to eligible 11-12 year-old patients. Only 75% of providers strongly disagreed with the statement 'vaccinating adolescents against HPV increases the likelihood of unprotected sex'. Even though 178/190 (94%) and 164/188 (87%) of surveyed family medicine providers reported recommending that their pregnant patients receive influenza vaccine and Tdap vaccine, respectively, only 134/185 (72%) routinely do so in their office., Conclusion: Most family medicine providers self-report always recommending standard pediatric vaccines, however only a minority are following ACIP recommendations. Educational sessions to update family medicine providers on ACIP recommendations and address individual provider concerns may improve provider vaccine confidence and uptake of vaccines by their patients.

Background: Nasopharyngeal pneumococcal carriage rates among HIV-infected adults has not been described since conjugate pneumococcal vaccine-13 (PCV13) was added to the universal infant and childhood vaccination schedule in 2010., Methods: HIV-infected adults presenting for routine health care visits to the Designated AIDS Center in Syracuse, NY between December 2013 and June 2015 were eligible for enrollment. Demographic, medical, and social history were recorded after obtaining informed consent. Nasopharyngeal samples were collected and cultured for the presence of Streptococcus pneumoniae using standard microbiologic techniques. Antibiotic susceptibility testing was performed using E-test→., Results: 707 nasopharyngeal samples were collected from 414 HIV-infected adults. 18 samples were culture positive for S. pneumoniae; 1 (6%) isolate was of vaccine-type, 9 (50%) were non-vaccine types, and 8 (44%) were non-typeable. The 18 isolates were recovered from 15 different patients (4% of those enrolled). Three patients were culture positive for pneumococcus isolated from 2 consecutive samples, with non-typeable pneumococci identified consecutively from 2 patients and serotype 35B identified consecutively from 1 patient. The most commonly identified non-vaccine serotypes were 35B and 15B/C. Identified pneumococci were penicillin and cefotaxime susceptible., Conclusion: Four percent of HIV-infected adults in our study population were colonized with S. pneumoniae. The non-vaccine serotypes 35B and 15B/C predominated.

Objective: To describe vaccine attitudes among pediatric healthcare providers attending immunization conferences., Study Design: Attendees of 5 American Academy of Pediatrics (AAP)-sponsored vaccine conferences held between June and November 2013 anonymously completed a questionnaire assessing vaccine attitudes and practices prior to the opening of educational sessions. Pearson's chi-square tests and Fisher's exact tests were used to analyze associations between vaccine attitudes, vaccine practices and provider characteristics., Results: 680 providers attending AAP-sponsored vaccine conferences were included. 661/666 (99%) enrolled providers state they routinely recommend standard pediatric vaccines, yet, 30 (5%) state that they do not routinely recommend influenza and/or human papillomavirus (HPV) vaccines. These providers expressed vaccine safety (87/680 (13%)) and efficacy (21/680 (31%)) concerns and stated belief in vaccine misperceptions: vaccine causes autism (34/668, 5%), multiple vaccines at a single visit reduces vaccine efficacy (43/680, 6%) or overwhelms the immune system (63/680, 9%), and administering HPV vaccine will increase the likelihood of unprotected adolescent sexual activity (29/680, 4%). Six percent of providers who do not routinely recommend all pediatric vaccines correctly identified themselves as vaccine hesitant., Conclusion: Vaccine hesitancy is under-recognized among pediatric providers attending AAP-sponsored immunization conferences. Educational interventions tailored to address provider vaccine concerns are needed to improve provider vaccine confidence., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chronic meningococcemia is a rare diagnosis seen in patients with recurrent fever and rash. We describe a case of chronic meningococcemia in a teenage girl who presented with a recurrent painful rash, without fever, over a period of 8 weeks.

Unlabelled: To determine factors associated with obstetric provider recommendation of pertussis vaccine (Tdap) to their pregnant patients following the Advisory Committee on Immunization Practices (ACIP) recommendation that Tdap be given in the third trimester of each pregnancy. Obstetric providers across New York State anonymously completed a standard set of questions to assess vaccine recommendation knowledge and practice., Statistical Analysis: Descriptive statistical methods were used to define provider characteristics, knowledge and vaccine practices. Factors associated with recommendation were analyzed using odds ratios. 133 obstetric providers were included in the study. 11% and 13% expressed concern with pertussis vaccine safety and efficacy, respectively, in pregnant women. 92% of obstetric providers stated that they knew ACIP recommendations for Tdap during pregnancy, 80% recommended Tdap to all eligible patients, but only 67% provided Tdap vaccine in their office. Provider knowledge of recommendation (OR 23.33), routine provider recommendation of influenza vaccine (OR 12.5), and administration of pertussis vaccine in the office (OR 7.01) were all factors strongly associated with routine provider recommendation of Tdap vaccine to eligible pregnant women (P < 0.05). Providers expressed concerns with cost of Tdap, the need to administer Tdap with each pregnancy, vaccine safety, low incidence of pertussis in the area, and administration of pertussis vaccine at the hospital after delivery. Educational programs are needed to improve provider vaccine confidence and recommendation.

Background: Pertussis is a highly contagious vaccine preventable disease resulting in significant infant morbidity and mortality. Despite the recommendations for pertussis vaccine (Tdap) in adults, coverage rates in this age group remain suboptimal. We sought to determine factors associated with Tdap receipt among adults with children in the household who live in central New York., Methods: The study team surveyed Tdap immunization status of adults who accessed medical services for their children provided by Golisano Children's Hospital, Syracuse, New York. Adults who did not know their Tdap vaccine status were excluded. Each participant was asked a standard set of questions to determine factors associated with Tdap receipt. Logistic regression was used to calculate simple and adjusted odds ratios for Tdap receipt in relation to adults' demographic characteristics, knowledge of Tdap and physician recommendations., Results: Eight hundred twenty four participants were included in this study; 34% had received Tdap in the past 5 years; 58% reported that their provider or child's pediatrician recommended adult Tdap vaccination. Tdap receipt was associated with knowing the symptoms of pertussis infection, female gender, younger age, and provider recommendation (p<0.05). Participants whose provider recommended Tdap vaccine were 24.6 times more likely to receive vaccine when compared to those whose providers did not recommend vaccine (95% CI: 16.3, 37.2, p<0.05)., Conclusion: Tdap coverage rates are low among this study population, with provider recommendation most strongly associated with Tdap receipt. Future steps to improve vaccine coverage should include both increasing community awareness and determining barriers to provider recommendation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Influenza vaccine (IV) coverage rates remain suboptimal among US adults. Socioeconomic disparities exist in IV coverage. We describe influenza vaccine attitudes among a low-income community in central New York., Methods: Adults attending a Salvation Army function during December 2012 were surveyed regarding IV including their intention to be immunized. On-site IV was offered to eligible participants., Results: The 1041 participants included Whites (non-Hispanics), African Americans, Hispanics, Native Americans, and multi-racial ethnicities. At time of enrollment, 386 (37%) participants had already received 2012-13 IV. Of the 655 unimmunized participants, 299 (46%) stated intent to receive IV, evenly distributed by age, gender, and ethnicity. Of the 312 participants who declined IV, 46% did so because of IV misperceptions. Of the 299 participants who intended to receive vaccine but had not yet done so, 284 (95%) stated the reason for delay was difficult access to vaccine. Intent to receive vaccine was strongly associated with the belief that IV is safe and/or effective (P < 0.05)., Conclusion: IV misperceptions regarding IV efficacy and safety result in suboptimal vaccine uptake in this low-income community, regardless of age, gender, or ethnicity.

Background: Using a murine model of parainfluenza virus infection (mPIV1 or Sendai virus; SeV), we compared the inflammatory responses to lethal and sub-lethal infections in inbred DBA/2 mice., Methods: Mice were intranasally inoculated with either 1.6×10(3) or 1.6×10(5) infectious units (IU) of SeV or diluent control. Clinical data including daily weights, oxygen saturation, and lung function via whole body plethysmography were collected on days 0, 3-7, and 9-14. Clarified whole lung homogenates were evaluated for inflammatory markers by enzyme-linked immunoassay (ELISA). Data were analyzed using ANOVA or Student t-tests, as appropriate., Results: Mice inoculated with 1.6×10(5) IU of SeV developed a lethal infection with 100% mortality by day 7, while mice inoculated with 1.6×10(3) IU developed a clinically significant infection, with universal weight loss but only 32% mortality. Interestingly, peak virus recovery from the lungs of mice inoculated with 1.6×10(5) IU of SeV did not differ substantially from that detected in mice that received the 100-fold lower inoculum. In contrast, concentrations of CCL5 (RANTES), CCL11 (eotaxin), interferon-γ, CXCL10 (IP-10), and CCL3 (MIP-1α) were significantly higher in lung tissue homogenates from mice inoculated with 1.6×105 IU (p < 0.05). In the lethal infection, levels of CCL11, interferon- γ and CCL3 all correlated strongly with disease severity., Conclusion: We observed that severity of SeV-infection in DBA/2 mice was not associated with virus recovery but rather with the levels of proinflammatory cytokines, specifically CCL11, interferon- γ and CCL3, detected in lung tissue in response to SeV infection.

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

Objective: We partnered with the Salvation Army to educate resource-poor families regarding childhood immunizations in an effort to improve vaccine coverage rates., Methods: Eligibility for enrollment included children of families presenting at registration for our Salvation Army holiday gift program, available to families with an annual income <150% of federal poverty guidelines. Parents completed a questionnaire, were provided each child's vaccination status as documented in the New York State Immunization Information System, and interacted with the study team to address immunization-related concerns. Missed vaccines were identified and parents were directed to their child's medical home for necessary immunizations. Vaccine coverage was ascertained via the New York State Immunization Information System every 6 to 8 weeks with telephone follow-up for children who remained delayed. The McNemar test and standard 2-proportion comparison were used to determine confidence intervals when analyzing matched or independent data, respectively., Results: A total of 1531 children were enrolled; 416 (28%) of the 1477 children with accurate immunization records were vaccine complete. When we excluded influenza vaccine, 1034 (70%) of children had received all other recommended vaccines. Nine months later, vaccine completion rates increased from 28% to 45%, largely because of improvements in influenza vaccination rates, which increased by 17% (confidence interval [CI] 15.5-19.5), a significant improvement over county (8%, 95% CI 7.4-8.1) and statewide (5%, 95% CI 4.7-4.8) rates during the same period., Conclusions: Immunization rates in poor children are suboptimal. Partnering with community-based organizations to address parental concerns, provide education, and perform follow-up was effective in improving immunization rates, particularly for influenza vaccine.

IL-21 is a cytokine with pleiotropic actions, promoting terminal differentiation of B cells, increased Ig production, and the development of Th17 and T follicular helper cells. IL-21 is also implicated in the development of autoimmune disease and has antitumor activity. In this study, we investigated the role of IL-21 in host defense to pneumonia virus of mice (PVM), which initiates an infection in mice resembling that of respiratory syncytial virus disease in humans. We found that PVM-infected mice expressed IL-21 in lung CD4(+) T cells. Following infection, Il21r(-/-) mice exhibited less lung infiltration by neutrophils than did wild-type (WT) mice and correspondingly had lower levels of the chemokine CXCL1 in bronchoalveolar lavage fluid and lung parenchyma. CD8(+), CD4(+), and γδ T cell numbers were also lower in the lungs of PVM-infected Il21r(-/-) mice than in infected WT mice, with normal Th17 cytokines but diminished IL-6 production in PVM-infected Il21r(-/-) mice. Strikingly, Il21r(-/-) mice had enhanced survival following PVM infection, and moreover, treatment of WT mice with soluble IL-21R-Fc fusion protein enhanced their survival. These data reveal that IL-21 promotes the pathogenic inflammatory effect of PVM and indicate that manipulating IL-21 signaling may represent an immunomodulatory strategy for controlling PVM and potentially other respiratory virus infections.

Background: Respiratory infections are a leading cause of pediatric hospitalizations. This study investigated whether virus-virus or virus-Bordetella co-infections are more frequent or more severe than previously recognized., Methods: This is a 3-year prospective study of children younger than 24 months hospitalized with a febrile respiratory illness. Viral pathogens were detected using multiplex polymerase chain reaction (PCR), enzyme-linked immunoassays, and/or viral cultures from nasopharyngeal samples. Bordetella infections were detected by PCR., Results: A total of 201 patients were enrolled. Respiratory viruses were detected in 187 (93%) patients, with 52 (28%) multipathogen infections. The most common viruses detected were respiratory syncytial virus and rhinovirus/enterovirus. There were no differences in illness severity when comparing patients infected with one pathogen and those with multipathogen infection., Conclusion: Virus co-infection in young children hospitalized with an acute febrile respiratory infection is common but does not appear to be associated with illness severity.

Background: The innate immune responses of neonates differ dramatically from those of adults. Here we examine the acute inflammatory responses of neonatal and weanling mice infected with pneumonia virus of mice (PVM), a rodent pathogen (family Paramyxoviridae, genus Pneumovirus) that replicates the sequelae of severe respiratory syncytial virus infection., Results: We demonstrate that virus replication proceeds indistinguishably in all age groups (inoculated at 1, 2, 3 and 4 weeks of age), although inflammatory responses vary in extent and character. Some of the biochemical mediators detected varied minimally with age at inoculation. Most of the mediators evaluated demonstrated elevated expression over baseline correlating directly with age at the time of virus inoculation. Among the latter group are CCL2, CCL3, and IFN-γ, all cytokines previously associated with PVM-induced inflammatory pathology in mature mice. Likewise, we detect neutrophil recruitment to lung tissue in all age groups, but recruitment is most pronounced among the older (3 - 4 week old) mice. Interestingly, all mice exhibit failure to thrive, lagging in expected weight gain for given age, including the youngest mice that present little overt evidence of inflammation., Conclusions: Our findings among the youngest mice may explain in part the phenomenon of atypical or minimally symptomatic respiratory infections in human neonates, which may be explored further with this infection model.

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

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

Background: We have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice., Results: In this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNgamma). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNgamma receptor gene-deleted mice, although neutrophils from IFNgammaR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNgamma. Interestingly, although supplemental IFNgamma restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNgamma -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-gamma together are sufficient to promote neutrophil recruitment but not pathologic activation., Conclusion: Our findings reveal a heretofore unrecognized hierarchical interaction between the IFNgamma and CCL3, which demonstrate that IFNgamma is crucial for CCL3-mediated neutrophil recruitment in vivo.

Immune responses to virus infection undergo significant change as part of the aging process. Here we examine the inflammatory responses of older, but otherwise immunologically naive mice to infection with pneumonia virus of mice (PVM). Although we see no changes in the extent or kinetics of virus replication, we observe diminished local production of inflammatory mediators, including MIP-1alpha, JE/MCP-1, IFN-gamma and IFN-gamma-induced MIG and IP-10, and interleukins (IL)-6 and IL-17. Levels of KC and IL-1alpha remained unchanged. Age-dependent diminished production of proinflammatory mediators was associated with diminished recruitment of granulocytes and reduced severity of clinical responses, including weight loss and respiratory dysfunction. The differences observed when comparing these results to those reported among elderly human subjects may be related to the specific extent of aging and its impact on biochemical and cellular inflammatory responses and/or the role of lifetime virus re-exposure on the clinical outcome from acute pneumovirus disease.

Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-alpha (TNF-alpha), interferon-beta (IFN-beta), macrophage inflammatory proteins 1alpha and 1beta (MIP-1alpha and MIP-1beta) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems.

We explore relationships linking clinical symptoms, respiratory dysfunction, and local production of proinflammatory chemokines in the pneumonia virus of mice (PVM) model of viral bronchiolitis. With a reduced inoculum of this natural rodent pathogen, we observe virus clearance by day 9, while clinical symptoms and respiratory dysfunction persist through days 14 and 17 postinoculation, respectively. Via microarray and ELISA, we identify expression profiles of proinflammatory mediators MIP-1alpha, MCP-1, and MIP-2 that correlate with persistent respiratory dysfunction. MIP-1alpha is localized in bronchial epithelium, which is also the major site of PVM replication. Interferon-gamma was detected in lung tissue, but at levels that do not correlate with respiratory dysfunction. Taken together, we present a modification of our pneumovirus infection model that results in improved survival and data that stand in support of a connection between local production of specific mediators and persistent respiratory dysfunction in the setting of acute viral bronchiolitis.

In this work we have evaluated the clinical responses of pneumovirus-infected mice to combination therapy with the antiviral agent, ribavirin, and the CysLT1 cysteinyl leukotriene receptor antagonist, montelukast. We observed substantial virus replication in our mouse model of pneumovirus infection and significant accumulation of cysteinyl leukotrienes in lung tissue, the latter detected at levels that correlate directly with granulocyte recruitment to the airways. While administration of the nucleoside analog, ribavirin, reduced virus replication approximately 2,000-fold, the clinical outcomes as measured by morbidity and mortality, in response to ribavirin monotherapy were indistinguishable from those of the no-treatment controls. Similarly, montelukast therapy alone did not reduce granulocyte recruitment nor did it improve the clinical outcome. However, combined therapy with ribavirin and montelukast resulted in a significant reduction in morbidity and a substantial reduction in mortality (50% survival at t = 14 days and onward, compared to 10-20% survival in response to montelukast alone or to ribavirin alone, respectively, p < 0.01). These findings define further the independent contributions made by virus replication and by the ensuing inflammatory response to the detrimental sequelae of pneumovirus infection in vivo.

Pneumonia virus of mice (PVM; family Paramyxoviridae) is a natural pathogen of rodents that reproduces important clinical features of severe respiratory syncytial virus infection in humans. As anticipated, PVM infection induces transcription of IFN antiviral response genes preferentially in wild-type over IFN-alphabetaR gene-deleted (IFN-alphabetaR-/-) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-gamma-inducible protein-10, and TLR-3 in lung tissue of IFN-alphabetaR-/- mice when compared with wild type. No differential expression of chemokines MIP-1alpha or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-alphabetaR-/- mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-alphabetaR-/- mice (50% survival at 10.8 +/- 0.6 days vs the wild type at 9.0 +/- 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-alphabetaR-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo.

Pneumonia virus of mice (PVM) is the first infection model that replicates features of severe human respiratory syncytial virus (hRSV) disease in the mouse. The PVM model has highlighted the importance of inflammation to the pathogenesis of severe disease, demonstrating that the inflammatory response remains active and acute even when virus replication ceases in response to appropriate antiviral therapy. The fact that the inflammatory response continues and is not completely linked to ongoing virus replication indicates the need for concurrent anti-inflammatory or, ideally, specific immunomodulatory therapy. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and its receptor, CC chemokine receptor 1 (CCR1), have been identified as crucial to the inflammatory response to PVM and hRSV and thus as elements to exploit for potential immunomodulatory control. Biochemical blockade of MIP-1alpha signaling with the CCR1 antagonist met-RANTES prevents the inflammatory response to PVM and results in reduced morbidity and mortality when administered in conjunction with the antiviral agent ribavirin. Ongoing exploration into the biology of PVM infection will identify other pathways and targets to be exploited for immunomodulatory control of hRSV and related severe respiratory virus infections.

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

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

The mouse eosinophil-associated ribonucleases (mEars) are species specific, divergent orthologs of the human antiviral RNase A ribonucleases, eosinophil-derived neurotoxin (RNase 2) and eosinophil cationic protein (RNase 3). We show here that mEar 2 is also an antiviral ribonuclease, as micromolar concentrations promote a approximately sixfold reduction in the infectivity of pneumonia virus of mice (PVM) for target respiratory epithelial cells in vitro. Although initially identified as a component of eosinophilic leukocytes, mEar 2 mRNA and protein were also detected in lung tissue accompanied by enzymatically active mEar 2 in bronchoalveolar lavage fluid (BALF). At t=3 days post-inoculation with PVM (strain J3666), we observed the characteristic inflammatory response accompanied by diminished expression of total mEar mRNA and protein in lung tissue and a corresponding fivefold drop in ribonuclease activity in BALF. No change in mEar expression was observed in response to infection with PVM strain 15, a replication-competent strain of PVM that does not elicit a cellular inflammatory response. However, mEar expression is not directly dependent on inflammation per se, as diminished expression of mEar mRNA and BAL ribonuclease activity were also observed in PVM-infected, inflammation-deficient, MIP-1alpha -/- mice. We propose that this mechanism may represent a novel virus-mediated evasion strategy, with a mechanism that is linked in some fashion to virus-specific pathogenicity.

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

Nitric oxide (NO) is generated by recruited inflammatory cells and by pulmonary epithelial cells in response to respiratory virus infection, although the relative antiviral efficacy of NO from each of these sources had not been clarified. To compare the direct, antiviral potency of NO from an exogenous source with that generated by target epithelial cells in situ, we transduced HEp-2 epithelial cells with the retroviral construct, pMFGS-NOS and cloned transductant lines that generated NO constitutively. We found that NO-producing HEp-2 cells could be infected with RSV, but the titer correlated inversely with NO production, an effect that was reversed by the NOS inhibitor, NG-methyl-L-arginine (NGMMA). Our results demonstrate that NO has significant direct antiviral activity against RSV, and interestingly, that the inhibitory effect is more potent in the presence of continuous, endogenous NO production than in response to NO from an exogenous source.

Pneumonia virus of mice (PVM; Paramyxoviridae, subfamily Pneumovirinae) is an important pathogen for the study of physiologically relevant acute inflammatory responses in rodent hosts. In contrast to the severe symptomatology observed in response to infection with PVM strain J3666, infection with strain 15 resulted in few clinical symptoms, limited cellular inflammatory response, and no production of macrophage inflammatory protein-1alpha or monocyte chemoattractant peptide (MCP)-1. Microarray analysis of transcripts from lung tissue indicates that PVM J3666 infection promotes up-regulation of specific proinflammatory genes, most notably interferon (IFN)-1beta, IFN response genes, and chemokines MCP-1, MCP-3, RANTES (regulated on activation, normally T cell-expressed and secreted), and eotaxin. Of these, only RANTES expression increased in response to infection with strain 15, with no increased expression of IFN or IFN response genes, despite ongoing viral replication. These results suggest that pneumovirus replication alone is insufficient to promote antiviral inflammation and that evaluation of the more divergent strain-specific pneumovirus proteins may provide some intriguing leads toward the molecular basis of this differential response.

The biology of the eosinophilic leukocyte-development, recruitment, and prolonged existence in somatic tissues-has been linked almost invariably to the actions of the "eosinophil" cytokine, interleukin-5 (IL-5). Here we demonstrate that pulmonary eosinophilia can occur in the absence of IL-5, as morphologically normal eosinophils are recruited to the lungs of virus-infected IL-5 -/- mice with kinetics and sequelae that are indistinguishable from those of their IL-5 +/+ counterparts. We conclude that pulmonary eosinophilia observed in response to primary paramyxovirus infection occurs via mechanisms that are distinct from those involved in eosinophil responses to allergens and in asthma. Furthermore, the presence of functional eosinophils in IL-5 -/- mice suggests the possibility of developmentally distinct subsets of what has been presumed to be a homogeneous leukocyte population.

The use of glucocorticoids for the treatment of symptoms associated with respiratory syncytial virus (RSV) infection has been questioned. To evaluate the sequelae of glucocorticoid administration in the setting of pneumovirus infection in vivo, hydrocortisone was administered to mice infected with pneumonia virus of mice (PVM), a pneumovirus and natural rodent pathogen that is closely related to RSV and replicates the signs and symptoms of severe human RSV infection. Results showed that hydrocortisone spared the pulmonary neutrophilia but resulted in ablation of the pulmonary eosinophilia, despite continued production of the relevant chemoattractant, macrophage inflammatory protein-1alpha. Hydrocortisone also led to diminished production of inducible nitric oxide synthase and accumulation of reactive nitrogen species in lung tissue and bronchoalveolar lavage fluid and diminished lymphocyte recruitment. PVM-infected mice responded to hydrocortisone with enhanced viral replication and accelerated mortality. These results suggest several mechanisms to explain why glucocorticoid therapy may be of limited benefit in the overall picture of pneumovirus infection.

In this work we continue our study of the biochemical responses of respiratory epithelial cells to infection with human paramyxovirus pathogens. In our earlier studies, we detected elevated concentrations of the proinflammatory chemokines MIP-1alpha and IL-8 in upper and lower respiratory tract secretions from patients infected with respiratory syncytial virus (RSV). Here we demonstrate the same trend for individuals infected with parainfluenza virus (PIV), with elevated concentrations of MIP-1alpha and IL-8 (means of 309 +/- 51 and 2280 +/- 440 pg/ml/mg protein, respectively) detected in nasal wash samples from 17 patients with culture-positive PIV. Similar to our findings with RSV, cells of the HEp-2 epithelial line and primary cultures of human bronchial epithelial cells respond to PIV infection with production and release of both MIP-1alpha and IL-8. Addition of the glucocorticoid anti-inflammatory agent hydrocortisone (200-1000 ng/ml) attenuated the production of MIP-1alpha and IL-8 in PIV-infected cells while having minimal to no effect on the production of these mediators from cells infected with RSV. Neither virus infection resulted in a change in the total cellular concentration of glucocorticoid receptors, nor did hydrocortisone exert any differential effect on viral replication. As repression of chemokine production by epithelial cells is likely to result in diminished recruitment of proinflammatory leukocytes, these results may explain in part why glucocorticoid therapy reduces the symptoms associated with acute PIV infection, but have little to no effect in the overall outcome in the case of RSV.

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

The intent of this study was to compare the cellular and biochemical inflammatory responses of mice infected with the paramyxovirus pathogens respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM). Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of the human disease, as a limited respiratory infection can be established via intranasal inoculation of virus at high titer. In earlier work, we found that acute infection with the natural rodent pathogen, PVM, elicited a rapid and sustained pulmonary inflammatory response (peak, 1.7 x 10(6) leukocytes/ml BAL fluid) that was dependent on both local production of MIP-1alpha and signaling via its receptor, CCR1. We find here that MIP-1alpha is also produced in response to RSV, although relatively few leukocytes (<200 ml BAL fluid) are recruited to the lungs in response. Further experiments with CCR1-deficient mice confirm the finding that although MIP-1alpha is produced in response to RSV infection, leukocytes do not respond via this pathway. Among the explanations for these findings, we propose that there are other, as yet to be identified proinflammatory mediators elicited in response to PVM (but not in response to RSV) that serve to prime the leukocytes in vivo, thus enabling them to respond to MIP-1alpha signaling via CCR1. Furthermore, the differences in disease pathogenesis seen in response to each of these pneumovirus infections in mice raise questions regarding the extent to which primary RSV infection in mice can be used as a model of primary RSV infection in humans., (Copyright 2000 Academic Press.)

We used differential display to detect enhanced expression of an mRNA fragment encoding cytokeratin 17 (Ck-17) in respiratory syncytial virus (RSV)-infected epithelial cells. Expression increased 12-fold by 96 h after infection but remained unchanged in cells challenged with virus in the presence of neutralizing anti-RSV fusion protein antibody. Immunoblots of RSV-infected cell lysates probed with an anti-keratin antibody demonstrated stable expression of total cytokeratins over time. When probed with an anti-Ck-17 monoclonal antibody, Ck-17 was first detected at 4 days after infection. In situ staining demonstrated that Ck-17 expression localized to regions of syncytia formation. Expression of Ck-17 mRNA also increased in response to intracellular RSV-F protein in the absence of active RSV infection. No increase in Ck-17 mRNA expression and no syncytia were observed in RSV-infected cells grown in the presence of the NF-kappaB inhibitor gliotoxin. These results suggest that RSV-induced transcriptional activation of the Ck-17 gene is dependent on an NF-kappaB-associated signaling pathway.

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1 alpha (MIP-1 alpha). Here we examine the role of MIP-1 alpha in the pathogenesis of this disease using mice deficient in MIP-1 alpha or its receptor, CCR1. The inflammatory response to PVM in MIP-1 alpha-deficient mice was minimal, with approximately 10-60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1 alpha-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1 alpha/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.

Human eosinophils secrete two distinct ribonucleases that have antiviral activity against pathogens of the family Paramyxoviridae. To examine the role of eosinophils and their ribonucleases in host defense against paramyxovirus pathogens in vivo, we have developed a mouse model involving a viral pathogen that naturally targets a rodent host. In this work we describe infection of Balb/c mice with pneumonia virus of mice (PVM, strain J3666), a paramyxovirus pathogen found frequently among rodent populations. We show here that pulmonary eosinophilia is an immediate response to infection with PVM, with bronchoalveolar lavage fluid containing 12-14% eosinophils obtained as early as day 3 postinoculation. Infection is accompanied by the production of macrophage inflammatory protein-1-alpha (MIP-1alpha), a chemokine that has been associated with the pulmonary eosinophilia observed in response to respiratory syncytial virus infection in humans and with enhanced clearance of influenza virus in mice. Interestingly, we observed no changes in expression of the chemoattractants eotaxin and RANTES in response to PVM infection, and interleukin-5 remained undetectable throughout. These responses-clinical pathology, viral recovery, pulmonary eosinophilia, and production of MIP-1alpha-will provide a means for exploring the role of eosinophils, eosinophil secretory ribonucleases, and eosinophil chemoattractants in host defense against PVM and related paramyxovirus pathogens in vivo., (Copyright 2000 Academic Press.)

By means of differential display reverse-transcriptase polymerase chain reaction, increased expression of the mRNA encoding the anti-apoptosis gene IEX-1L was found in respiratory epithelial cells infected with respiratory syncytial virus (RSV). IEX-1L mRNA expression increased 5-7-fold in RSV-infected cells at 72 h after infection but remained unchanged in cells exposed to irradiated, replication-incompetent RSV. Because IEX-1L is reported to protect cells from apoptosis induced by tumor necrosis factor (TNF)-alpha, the effect of TNF-alpha on epithelial cell apoptosis in the context of RSV infection was determined. Epithelial cells were exposed to vehicle, RSV, or irradiated RSV for 72 h, and then TNF-alpha was added to appropriate cultures. Cytochemical staining of cellular DNA with 4,6-diamidino-2-phenylindole demonstrated TNF-alpha-induced apoptosis in 23.4% of control cells but only 5% of RSV-infected cells. These data show that RSV infection protects epithelial cells from TNF-alpha-induced apoptosis and that this effect is temporally associated with IEX-1L gene expression.

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

Characterization of chemokine expression patterns in virus-infected epithelial cells provides important clues to the pathophysiology of such infections. The aim of this study was to determine the chemokine response pattern of respiratory epithelium when infected with respiratory syncytial virus (RSV). Macrophage inflammatory protein-1-alpha (MIP-1-alpha), interleukin-8 (IL-8), and RANTES concentrations were measured from RSV-infected HEp-2, MRC-5, and WI-38 cell culture supernatants daily following infection. Additionally, MIP-1-alpha, IL-8, and RANTES concentrations were measured from lower respiratory secretions obtained from 10 intubated infants (0-24 mo) with RSV bronchiolitis, and from 10 control subjects. Our results indicate that respiratory epithelial cells respond to RSV infection by producing MIP-1-alpha, IL-8, and RANTES. Production of MIP-1-alpha required ongoing viral replication, whereas RANTES and IL-8 could be elicited by inactivated forms of the virus. MIP-1-alpha, RANTES, and IL-8 were also present in lower airway secretions obtained from patients with RSV bronchiolitis. Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), the eosinophil secretory ribonucleases, were detected in lower airway secretions from RSV-infected patients; ECP concentrations correlated with MIP-1-alpha concentrations (r = 0.93). We conclude that MIP-1-alpha is present in the lower airways during severe RSV disease. The correlation between MIP-1-alpha and ECP concentrations suggests a role for eosinophil degranulation products in the pathogenesis of RSV bronchiolitis.

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

Macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were measured by enzyme-linked immunosorbent assay (ELISA) from virus-infected respiratory cell culture supernatants and from 100 nasal wash samples obtained from patients aged 8 d to 10 yr. The results of the nasal wash samples were analyzed in relation to the etiology of the viral infection. In vitro, respiratory syncytial virus (RSV) induced the production of MIP-1alpha, while both RSV and adenovirus were associated with the production of RANTES. Both MIP-1alpha and RANTES were detected in nasopharyngeal secretions from pediatric patients with acute upper respiratory tract RSV, adenovirus, influenza, and parainfluenza virus infection (p<0.001 by Fisher's exact test). As both of these chemokines have potent effects on the recruitment and degranulation of eosinophils and basophils, further understanding of their role in upper respiratory tract infections may provide valuable insights into the immunopathogenesis of respiratory viral infections.

We have demonstrated that the human eosinophil-derived neurotoxin (EDN, RNase 2), a rapidly evolving secretory protein derived from eosinophilic leukocytes, mediates the ribonucleolytic destruction of extracellular virions of the single-stranded RNA virus respiratory syncytial virus (RSV). While RNase activity is crucial to antiviral activity, it is clearly not sufficient, as our results suggest that EDN has unique structural features apart from RNase activity that are necessary to promote antiviral activity. We demonstrate here that the interaction between EDN and extracellular virions of RSV is both saturatable and specific. Increasing concentrations of the antivirally inactivated, ribonucleolytically inactivated point mutant form of recombinant human EDN, rhEDNdK38, inhibits rhEDN's antiviral activity, while increasing concentrations of the related RNase, recombinant human RNase k6, have no effect whatsoever. Interestingly, acquisition of antiviral activity parallels the evolutionary development of the primate EDN lineage, having emerged some time after the divergence of the Old World from the New World monkeys. Using this information, we created ribonucleolytically active chimeras of human and New World monkey orthologs of EDN and, by evaluating their antiviral activity, we have identified an N-terminal segment of human EDN that contains one or more of the sequence elements that mediate its specific interaction with RSV.