Severe Respiratory Illness In Children Research Articles

EV-D68 virus-like particle vaccines elicit cross-clade neutralizing antibodies that inhibit infection and block dissemination.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4+ T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.

The national burden of influenza-like illness and severe respiratory illness overall and associated with nine respiratory viruses in South Africa, 2013-2015.

BackgroundEstimates of the disease burden associated with different respiratory viruses are severely limited in low‐ and middle‐income countries, especially in Africa.MethodsWe estimated age‐specific numbers and rates of medically and non‐medically attended influenza‐like illness (ILI) and severe respiratory illness (SRI) that were associated with influenza, respiratory syncytial virus (RSV), rhinovirus, human metapneumovirus, adenovirus, enterovirus and parainfluenza virus types 1–3 after adjusting for the attributable fraction (AF) of virus detection to illness in South Africa during 2013–2015. The base rates were estimated from five surveillance sites and extrapolated nationally.ResultsThe mean annual rates per 100,000 population were 51,383 and 4196 for ILI and SRI, respectively. Of these, 26% (for ILI) and 46% (for SRI) were medically attended. Among outpatients with ILI, rhinovirus had the highest AF‐adjusted rate (7221), followed by influenza (6443) and adenovirus (1364); whereas, among inpatients with SRI, rhinovirus had the highest AF‐adjusted rate (400), followed by RSV (247) and influenza (130). Rhinovirus (9424) and RSV (2026) had the highest AF‐adjusted rates among children aged <5 years with ILI or SRI, respectively, whereas rhinovirus (757) and influenza (306) had the highest AF‐adjusted rates among individuals aged ≥65 years with ILI or SRI, respectively.ConclusionsThere was a substantial burden of ILI and SRI in South Africa during 2013–2015. Rhinovirus and influenza had a prominent disease burden among patients with ILI. RSV and influenza were the most prominent causes of SRI in children and the elderly, respectively.

Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three‐dimensional reconstruction, we show that there is extensive helical ordering of the envelope‐associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub‐tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M‐dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M‐layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N‐RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

Enterovirus D68 outbreak detection through a syndromic disease epidemiology network

BackgroundIn 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). ObjectivesUsing the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. Study designPER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. ResultsThe final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. ConclusionsUsing PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories

982. Summer Outbreak of Severe RSV-B Disease, Minnesota, 2017

BackgroundRespiratory syncytial virus (RSV) causes seasonal outbreaks peaking from October to April in the United States. While symptoms are typically mild and limited to the upper respiratory tract in older children and adults, RSV can cause severe lower respiratory disease, hospitalization and death, particularly among younger children, the immunocompromised and the elderly. During summer 2017, the Minnesota Department of Health (MDH) received a report of a cluster of severe respiratory illness in children with RSV-B infection admitted at an urban county hospital.MethodsMDH conducts surveillance for RSV in Minneapolis-St. Paul with collection of respiratory specimens and clinical and demographic data. We compared characteristics of cases reported in summer 2017 with the previous 4 summer seasons. To understand the genetic relatedness among viruses, we performed complete genome sequencing from primary specimens using an Illumina MiSeq platform employing both a sequence-independent sequencing method and an RSV-specific, overlapping PCR-based approach.ResultsFrom May to September 2017, 58 cases of RSV (48 RSV-B) were reported to MDH compared with 18 (7 RSV-B) during the same period in 2016, 27 in 2015, and 29 in 2014. The median age and frequency of co-morbidities were similar across years; however, 50% required ICU admission in 2017 compared with 12% in preceding 3 years. The RSV-B genome was sequenced from 10 specimens from March to May 2016, 20 specimens from May to September 2017 and 30 specimens from October 2017-January 2018. Phylogenic analysis revealed that 15 cases from May to September. 2017 formed a unique clade distinct from the lineage encompassing 2016 cases from Minnesota and 35 representative complete RSV genomes in GenBank isolated from 6 continents over 13 years. From October 2017 to January 2018, we detected co-circulation of viruses from both lineages among older adults and children.ConclusionWe identified an outbreak of RSV-B associated with severe disease among urban Minnesota children during a time of expected low RSV circulation. Complete genome sequencing data suggested emergence of a new lineage distinct from viruses circulating in Minnesota during the previous season. Genomic characterization can provide useful insights into epidemiologic variations.Disclosures All authors: No reported disclosures.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

Human enterovirus D68 in clinical and sewage samples in Israel

BackgroundSince mid-August 2014, North America experienced a wide outbreak of Enterovirus D68 (EV-D68) associated with severe respiratory illness in children. Several other countries also reported cases of EV-D68 in 2014. ObjectivesThe aim of this study was to determine whether EV-D68 circulated in Israel in 2014, caused severe respiratory illness in children and was the causative agent of Acute Flaccid Paralysis. Study designArchived clinical respiratory samples from a cohort of 710 hospitalized pediatric patient’s (<10years old) with respiratory illness were screened for clade B specific EV-D68 by real-time PCR. The patients were seen at four medical centers covering the entire country between August and November 2014. We also evaluated 49 patient stool samples from 26 AFP cases during 2014 for presence of EV-D68. In addition, RNA from sewage samples collected throughout Israel during the same study period was also tested for EV-D68. Partial VP1 sequencing was performed on all positive samples. ResultsOf the 710 clinical samples evaluated, 7 (1%) were positive for EV-D68. Two patients were from the central part of Israel, while the rest was from the southern part. The majority of the patients did not have any underlying disease. Not only that, but, none of the 26 suspected AFP cases had EV-D68 nucleic acid in their stool samples. EV-D68 RNA was detected in 9 out of 93 sewage samples, mainly from Southern Israel. Sequence analysis of EV-D68 VP1 gene from both sewage and clinical samples indicated that the Israeli EV-D68 RNA belonged to Clade B which was genetically similar to 2014 circulating European and North American EV-D68 virus. ConclusionsEV-D68 circulated in Israel during the 2014 summer-fall season and caused hospitalization of a small percent of the patients with respiratory illness.

Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States.

In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans.

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

Pharmacists critical to managing antimicrobials, developing infection control procedures

Infectious diseases kill more people worldwide than any other single cause, according to the U.S. National Library of Medicine. With Ebola raising its ugly head, combined with the mysterious enterovirus D68 and the fact that we're heading into cold and influenza season, hospital pharmacists continue to play key roles in infectious disease control, antimicrobial stewardship programs, and managing antibiotics.

Marked difference between adults and children in Bordetella pertussis DNA load in nasopharyngeal swabs

Bordetella pertussis is the aetiologic agent of whooping cough, a common cause of severe respiratory illness in children and prolonged mild cough in adults. To understand some of the reasons for differences in clinical symptoms between adults and children, we measured B. pertussis DNA loads in nasopharyngeal swabs (NPS) from 19 adults and 40 children (including 14 infants) by quantitative IS481 real-time PCR. All cases had been pre-diagnosed with the B. pertussis-specific loop-mediated isothermal amplification method. The mean PCR threshold cycles for adult and child NPS were 34.9 and 27.1, respectively, indicating a significantly lower B. pertussis DNA load in adults than in children (p <0.001). Moreover, adults had very low DNA loads during both early and later stages of the disease. When corresponding bacterial loads in NPS were calculated for B. pertussis Tohama cells using a standard curve, the mean number of bacterial cells taken with a rayon-tipped swab from an adult, older child and infant was estimated to be 320 (95% CI 120–910), 2.1 × 104 (95% CI 5.3 × 103 to 8.3 × 104) and 1.1 × 106 cells (95% CI 1.2 × 105 to 8.9 × 106), respectively. This indicates that the B. pertussis load in NPS is closely correlated with patient age. Our observations suggest that adult pertussis is characterized by a lower bacterial load in the nasopharynx, resulting in milder symptoms and negative cultures.

Virus Respiratorios en Menores de Diez Años con Infección Respiratoria en el Hospital Militar Central de Bogotá 2000-2001

A study was carried out on children seeking medical care at the Hospital Militar (Hosmil) in Bogota due to acute respiratory infection; it was aimed at estimating the frequency of respiratory virus-associated acute respiratory disease (ARD) in children aged under 10. Three to five children aged less than 10 years old were selected every week from the hospital's emergency ward or ambulatory services over an 18-month period from March 2000 to September 2001. Nasopharyngeal samples were obtained from them and processed using indirect immunofluorescence. The relative frequency of respiratory syncytial virus (RSV), influenza, adenovirus and parainfluenza were calculated. Around 40% of the 139 children examined proved positive for at least one respiratory virus. RSV was the most frequently found virus (27% positivity: 38 patients) followed by influenza (5%: 7 patients) and adenovirus (3%: 4 patients). RSV circulation peaks coincided with peaks related to ARD and pneumonia in the hospital: it was found most frequently in children aged less than 3 and was present throughout the year. Respiratory viruses were an important cause of moderate to severe respiratory illness in children seeking health care at Hosmil; RSV was the leading agent amongst such viruses.

Outbreak of Human Adenovirus Type 3 Infection in a Pediatric Long-Term Care Facility--Illinois, 2005

Human adenovirus type 3 (HAdV-3) causes severe respiratory illness in children, but outbreaks in long-term care facilities have not been frequently reported. We describe an outbreak of HAdV-3 infection in a long-term care facility for children with severe neurologic impairment, where only 3 of 63 residents were ambulatory. A clinical case of HAdV-3 was defined as fever (temperature, > or = 38.0 degrees C) and either a worsening of respiratory symptoms or conjunctivitis in a resident, with illness onset from June through August 2005. We reviewed medical records; conducted surveillance for fever, conjunctivitis, and respiratory symptoms; and collected nasopharyngeal and conjunctival specimens from symptomatic residents. Specimens were cultured in HAdV-permissive cell lines or were analyzed by HAdV-specific polymerase chain reaction assay. Thirty-five (56%) of 63 residents had illnesses that met the case definition; 17 patients (49%) were admitted to intensive care units, and 2 (6%) died. Patients were hospitalized in the intensive care unit for a total of 233 patient-days. Illness onset dates ranged from 1 June through 24 August 2005. Thirty-two patients (91%) had respiratory infection, and 3 (9%) had conjunctivitis. HAdV was identified by culture or PCR in 20 patients. Nine isolates were characterized as HAdV-3 genome type a2. Considering the limited mobility of residents and their reliance on respiratory care, transmission of HAdV-3 infection during this outbreak likely occurred through respiratory care provided by staff. In environments where patients with susceptible underlying conditions reside, HAdV infection should be considered when patients are identified with worsening respiratory disease, and rapid diagnostic tests for HAdV infection should be readily available to help identify and curtail the spread of this pathogen.

Human Parainfluenza Virus 4 Outbreak and the Role of Diagnostic Tests

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.

Involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type 3: possible role of actin in other viruses.

Multifunctional involvement of actin microfilaments during viral infection has been documented in many studies. The molecular mechanism underlying this important host-virus interaction, however, remains poorly understood. We have investigated the role of actin microfilaments in the life cycle of human parainfluenza virus type 3 (HPIV3), a paramyxovirus that causes severe respiratory illness in children. In vitro transcription with purified viral ribonucleoprotein (RNP) complex showed a requirement of cellular actin, in the polymeric form, for mRNA synthesis in vitro. This was further confirmed by using recombinant actin, which interacted with the viral RNP and also activated mRNA synthesis in vitro. Consistent with the role of the polymeric form of actin, the actin microfilaments of the cytoskeletal framework participate in the virus replication in vivo. Biochemical and immunological analyses revealed the association of viral RNPs with cytoskeletal framework during early stages of infection, and involvement of these RNPs in the synthesis of mRNAs and genome-length RNA. Immunofluorescent labeling and confocal microscopy showed that the viral nucleocapsids colocalize with the actin microfilaments. Treatment of cells with cytochalasin D, which depolymerizes actin microfilaments, inhibited viral RNA synthesis and RNP accumulation. These data indicate that actin microfilaments play a critical role in HPIV3 life cycle, specifically at the level of viral transcription and replication. Involvement of the cytoskeletal framework in the life cycle of several viruses containing RNA and DNA genomes is reviewed.

Involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type 3: Possible role of actin in other viruses

Multifunctional involvement of actin microfilaments during viral infection has been documented in many studies. The molecular mechanism underlying this important host-virus interaction, however, remains poorly understood. We have investigated the role of actin microfilaments in the life cycle of human parainfluenza virus type 3 (HPIV3), a paramyxovirus that causes severe respiratory illness in children. In vitro transcription with purified viral ribonucleoprotein (RNP) complex showed a requirement of cellular actin, in the polymeric form, for mRNA synthesis in vitro. This was further confirmed by using recombinant actin, which interacted with the viral RNP and also activated mRNA synthesis in vitro. Consistent with the role of the polymeric form of actin, the actin microfilaments of the cytoskeletal framework participate in the virus replication in vivo. Biochemical and immunological analyses revealed the association of viral RNPs with cytoskeletal framework during early stages of infection, and involvement of these RNPs in the synthesis of mRNAs and genome-length RNA. Immunofluorescent labeling and confocal microscopy showed that the viral nucleocapsids colocalize with the actin microfilaments. Treatment of cells with cytochalasin D, which depolymerizes actin microfilaments, inhibited viral RNA synthesis and RNP accumulation. These data indicate that actin microfilaments play a critical role in HPIV3 life cycle, specifically at the level of viral transcription and replication. Involvement of the cytoskeletal framework in the life cycle of several viruses containing RNA and DNA genomes is reviewed. Microsc. Res. Tech. 47:114–123, 1999. © 1999 Wiley-Liss, Inc.