Respiratory Disease In Infants Research Articles

No detection of human bocavirus in amniotic fluid samples from fetuses with hydrops or isolated effusions

Background Human bocavirus (HBoV) is a recently identified parvovirus associated with respiratory disease in infants. Animal bocaviruses have been shown to cause intrauterine infection, fetal anasarca and abortion in late gestation. Objectives To investigate whether HBoV infection is associated with fetal hydrops, fetal anemia or isolated fetal effusions. Study design We determined the prevalence of HBoV and parvovirus B19 (B19) DNA in amniotic fluid samples from fetuses with hydrops, anemia or isolated effusions using different real-time PCR protocols, and the HBoV IgG and IgM positivity rate in pregnant women with fetal hydrops or normal ultrasound findings by a non-commercial virus-like particle-based enzyme immunoassay. Results None of 87 amniotic fluid samples tested was HBoV DNA positive. Twelve of 60 fetuses with hydrops or anemia were found B19 DNA positive. Anti-HBoV IgG antibodies were detected in 100% (19/19) and 94% (47/50) of serum samples from pregnant women with fetal hydrops and normal ultrasound findings, respectively. All serum samples were found negative for anti-HBoV IgM. Conclusion We suggest that HBoV is not a common cause of fetal hydrops, anemia or isolated effusions. This has to be confirmed by further studies of proven gestational HBoV infection.

Epitope-Specific Regulatory T cells Differentially Modulate the CD8 T Cell Response to Respiratory Syncytial Virus in Mice (45.29)

Abstract RSV infection is the leading cause of lower respiratory disease in infants and young children, accounting for >120,000 hospitalizations each year in the US alone. Although CD8 T cell effector function is key to virus clearance, CD4 T cells coordinate the immune response and can lead to immunopathology. Therefore, they play a central role in the immunity and immunopathogenesis of RSV infection. To facilitate in depth evaluation of RSV-induced CD4 T cell responses we identified two new I-Ab- restricted dominant CD4 T cell epitopes, M213-223 and M227-37, in RSV M and M2 that also contain well characterized CD8 T cell epitopes. CD4 T cells from lung and spleen of RSV-infected CB6F1 mice produce different patterns of IL-2, IFN-γ and other cytokines after peptide stimulation. Novel tetrameric peptide-I-Ab complexes identified 0.5% and 1.6% RSV M- and M2-specific CD4 T cells in lungs of the infected mice. Peptide-stimulated CD4 T cells produced different patterns of regulation on CD8 T cell responses to RSV M and M2 epitopes in vitro. The regulatory function of the CD4 T cell correlated with the level of FoxP3 expression in epitope-specific CD4 T cells Our study demonstrates that the biophysical properties of the epitope may dictate the functional properties of the CD4 T cell response. Understanding how epitope structure influences T cell function will provide new insight into viral pathogenesis and may improve vaccine design.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (P) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines.

Assessment and predictor determination of indoor aldehyde levels in Paris newborn babies’ homes

Exposure to indoor chemical air pollutants expected to be potentially involved in allergic respiratory diseases in infants is poorly documented. A specific environmental investigation included in a birth cohort study was carried out to first assess indoor airborne aldehyde levels, using passive devices and their variability within 1 year (1, 6, 9 and 12 months) in the bedroom of 196 Paris infants, and second, to identify predictors for aldehyde concentrations using interviewer administered questionnaires about housing factors. Comfort parameters and carbon dioxide levels were measured simultaneously. Aldehydes were detected in almost all dwellings and geometric mean levels (geometric standard deviation) at the first visit were respectively for formaldehyde, acetaldehyde, hexanal, and pentanal 19.4 (1.7) microg/m(3), 8.9 (1.8) microg/m(3), 25.3 (3.1) microg/m(3), 3.7 (2.3) microg/m(3), consistent with earlier published results. Generalized Estimating Equation multivariate analyses showed that, apart from comfort parameters, aeration and season, the main indoor aldehyde sources were either continuous (building materials and coverings especially when they were new) or discontinuous (smoking, use of air fresheners and cleaning products, DIY etc...). Finally, the data collected by questionnaires should be sufficient to enable us to classify each infant in our cohort study according to his/her degree of exposure to the main aldehydes. This analysis contributed to document indoor aldehyde levels in Parisian homes and to identify factors determining these levels. In the major part of newborn babies' homes, indoor formaldehyde levels were above the guideline value of 10 microg/m(3) proposed by the French Agency for Environmental and Occupational Health Safety for long-term exposure. Given this result, it is essential to study the health impact of exposure to aldehydes especially formaldehyde on the incidence of respiratory and allergic symptoms, particularly during the first months of life.

Morphogenesis of Coronavirus HCoV-NL63 in Cell Culture: A Transmission Electron Microscopic Study

NL63 (HCoV-NL63) is a recently discovered human coronavirus that causes respiratory disease in infants and young children. NL63 productively infects LLCMK2 cells and ciliated epithelial cells of human airway cell cultures. Transmission electron microscopic (TEM) studies of NL63 infected LLCMK2 cells revealed that virions are spherical, spiked, and range from 75 to 115 nm in diameter. Virus replication predominantly occurs on the rough endoplasmic reticulum (RER), both perinuclear and cytoplasmic, and the Golgi. Plasma membrane budding was occasionally observed. As virus production increased, aberrant viral forms appeared with greater frequency. Unusual inclusions were present in infected cells including tubular and laminated structures. Pleomorphic double membrane-bound vesicles (DMV), measuring roughly 140 to 210 nm in diameter, were observed. The virus was released via exocytosis and cell lysis. In summary, we report the key morphologic characteristics of NL63 infection observed by TEM analysis.

Pulmonary immunity and immunopathology: lessons from respiratory syncytial virus

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and is an important source of morbidity and mortality in the elderly and immunocompromised. This review will discuss the humoral and cellular adaptive immune responses to RSV infection and how these responses are shaped in the immature immune system of the infant and the aged environment of the elderly. Furthermore, we will provide an overview of our current understanding of the role the various arms of the adaptive immune response play in mediating the delicate balance between the successful elimination of the virus from the host and the induction of immunopathology. Efficacious immunization against RSV remains a high priority within the field and we will highlight recent advances made in vaccine design.

Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil

Respiratory syncytial virus (RSV) is well recognized as the most important pathogen causing acute respiratory disease in infants and young children, mainly in the form of bronchiolitis and pneumonia. Two major antigenic groups, A and B, have been identified; however, there is disagreement about the severity of the diseases caused by these two types. This study investigated a possible association between RSV groups and severity of disease. Reverse transcription-polymerase chain reaction was used to characterize 128 RSV nasopharyngeal specimens from children less than five years old experiencing acute respiratory disease. A total of 82 of 128 samples (64.1%) could be typed, and, of these, 78% were group A, and 22% were group B. Severity was measured by clinical evaluation associated with demographic factors: for RSV A-infected patients, 53.1% were hospitalized, whereas for RSV B patients, 27.8% were hospitalized (p = 0.07). Around 35.0% of the patients presented risk factors for severity (e.g., prematurity). For those without risk factors, the hospitalization occurred in 47.6% of patients infected with RSV A and in 18.2% infected with RSV B. There was a trend for RSV B infections to be milder than those of RSV A. Even though RSV A-infected patients, including cases without underlying condition and prematurity, were more likely to require hospitalization than those infected by RSV B, the disease severity could not to be attributed to the RSV groups.

Prevention of allergic respiratory disease in infants: current aspects and future perspectives

Primary and secondary prevention of severe atopic disease exemplified by atopic asthma represents an increasingly prominent focus of research in paediatric allergy. We review below the rationale for this approach and recent progress in development and testing of a range of potential preventive strategies. The principal areas reviewed relate to potential and currently available treatments targeting enhancement of overall immune functions, allergen-specific immunomodulation and respiratory viral infections, particularly during early childhood. The scientific rationale for prophylaxis of atopic diseases via early intervention strategies targeting young children is increasingly supported by findings from the experimental and clinical literature and significant progress may be expected in the mid-term future.

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.

Vaccination with recombinant modified vaccinia virus Ankara expressing bovine respiratory syncytial virus (bRSV) proteins protects calves against RSV challenge

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.

Risk factors for meconium aspiration in meconium stained amniotic fluid

SummaryMeconium aspiration syndrome (MAS) is a life-threatening respiratory disease in infants born through meconium stained amniotic fluid (MSAF). The purpose of this study was to determine risk factors for MAS in the newborns of mothers who had meconium stained amniotic fluid in labour. A retrospective study of all full-term pregnancies with MSAF from May 2003 to October 2004 was designed at a teaching hospital. Development of MAS was the primary outcome. Maternal details, mode of delivery and neonatal details (Apgar score, reassuring or non-reassuring fetal heart rate tracing and birth weight) were evaluated. During the study period, there were 2,603 deliveries of whom 302 (11.6%) had MSAF. MAS developed in 64 of these infants (21.1%). Compared with healthy neonates with MSAF, those with MAS had higher rate of non-reassuring fetal heart rate (FHR) tracing, thick meconium and Apgar score ≤5 at 5 min. The neonatal birth weight was lower in the MAS group, maternal age, parity, gestational age and mode of delivery were not significantly different in the two group. We found the severity of meconium, low Apgar score at 5 min and non-reassuring FHR tracing was associated with MAS in MSAF pregnancies.

Nebulized Hypertonic Saline Shortens Hospital Stay in Infants with Bronchiolitis

Bronchiolitis is a common and potentially serious viral respiratory disease in infants. Because pharmacologic therapies (e.g., steroids, β-agonists) are generally thought to be ineffective, treatment is largely supportive, consisting mainly of hydration and oxygen. This study evaluated a relatively novel treatment — nebulized hypertonic saline — which might …

Alkaline phosphatase isoenzymes in children with respiratory disease

Aim: To present the electrophoretic pattern of alkaline phosphatase (ALP) isoenzymes in serum of infants and children exhibiting increased total ALP catalytic activity in the course of acute respiratory disease. Subjects and methods: Results obtained in 21 children (17 of them infants), including 13 male and eight female children aged 2 months to 8 years, hospitalized for respiratory diseases are presented. Total ALP catalytic activity was determined and electrophoretic separation of ALP isoenzymes was performed in children's sera. Results: Increased total ALP catalytic activity (range, 528-5622 U/L) during hospital stay was recorded in eight (38.1%) children. A typical picture of be-nign transient hyperphosphatasemia (TH), which implies the occurrence of fast anodal fraction (faster than hepatic fraction) and near-cathode fraction (faster than bone fraction), was recorded in five children. The placental-like isoenzyme was detected in two children. Expression of bone fraction and placental-like fraction was recorded in a rachitic child. Prehepatic ALP was expressed in two children, and hepatic ALP isoenzyme in one child. Conclusion: Acute respiratory disease in infants and children may entail transient increase in the ALP catalytic activity with the occurrence of various isoenzyme bands such as fast anodal and near-cathode fraction (in TH), prehepatic fraction and placental-like fraction. TH is verified when total ALP activity has decreased and returned to reference intervals. In this case, no additional testing is required.

A serum-free Vero production platform for a chimeric virus vaccine candidate

MedImmune Vaccines has engineered a live, attenuated chimeric virus that could prevent infections caused by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV), causative agents of acute respiratory diseases in infants and young children. The work here details the development of a serum-free Vero cell culture production platform for this virus vaccine candidate. Efforts to identify critical process parameters and optimize culture conditions increased infectious virus titers by approximately 2 log(10) TCID(50)/ml over the original serum-free process. In particular, the addition of a chemically defined lipid concentrate to the pre-infection medium along with the shift to a lower post-infection cultivation temperature increased virus titers by almost 100-fold. This improved serum-free process achieved comparable virus titers to the serum-supplemented process, and demonstrated consistent results upon scale-up: Vero cultures in roller bottles, spinner flasks and bioreactors reproducibly generated maximum infectious virus titers of 8 log(10) TCID(50)/ml.

The Association of Respiratory Syncytial Virus Infection and Influenza with Emergency Admissions for Respiratory Disease in London: An Analysis of Routine Surveillance Data

The importance of respiratory syncytial virus (RSV) infection in adults is not well known, because laboratory testing for RSV infection is not routine. Both RSV infection and influenza are seasonally related, and it is difficult to disentangle one from the other and to disentangle infection from the season and the cold. Emergency hospitalizations for respiratory disease from April 1994 to March 2001 were analyzed in relation to surveillance data on RSV infection and influenza, using Poisson regression models adapted for time series and adjusted for season, outdoor temperature, and other covariates. Age-specific admission rates attributable to the viruses were also estimated. Most of the crude relationships of emergency admissions of patients with RSV infection were confounded by season and, to a lesser extent, by cold temperatures. After adjustment for all covariates, including influenza, a 10th-90th percentile increase in RSV counts (defined as the daily number of laboratory reports of RSV) was associated with a rate ratio of 1.36 (95% CI, 1.27-1.45) for emergency admissions for respiratory disease in infants. The rate of hospitalization attributable to RSV infection in children aged <1 year was 5 per 1000 infants per year. The association in people > or =65 years old was much smaller (rate ratio, 1.09; 95% CI, 1.04-1.14; attributable rate of hospitalization , 0.7 per 1000 population); but unlike the association in infants, this association became smaller as the lag (interval) between infection and hospital admission became shorter. Admission rates attributable to influenza were highest in the > or =65-year age group (1.1 per 1000 population), but it was very small at younger ages. RSV infection appears to be an important determinant of hospitalization in infants in this data set but appears less certain for older persons and requires further investigation.

779. Genetic Delivery of an Anti-RSV Antibody Protects Against Pulmonary Infection with RSV in Mice

Respiratory syncytial virus (RSV) is the leading cause of severe viral respiratory disease in infants and children. The morbidity is greatest in young infants, children with underlying risk factors such as prematurity and bronchopulmonary dysplasia, and the elderly. A safe and efficient active vaccine is not available. Passive immunization with the monoclonal antibody palivizumab, a humanized murine IgG1 antibody targeted against a neutralizing epitope of the F glycoprotein of RSV, diminishes disease severity in high risk populations and is routinely used to protect premature infants during the RSV season.

Genetic Variability and Molecular Evolution of the Human Respiratory Syncytial Virus Subgroup B Attachment G Protein

Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 x 10(-3) nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 x 10(-3) nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.

Acute and long-term effects of infection by the respiratory syncytial virus in children with congenital cardiac malformations

All newborn infants have limited pulmonary reserve compared with older children. This puts them at increased risk of respiratory complications, such as those associated with infection by the respiratory syncytial virus. Young children with congenital cardiac disease are particularly likely to suffer severe disease related to infection by the virus. In these children, the extreme vulnerability of the lung to pulmonary oedema is compounded by the additional burden caused by the respiratory syncytial virus. In addition to the well-documented acute pulmonary effects of infection with the respiratory syncytial virus, there may also be consequent long-term respiratory morbidity. Clinical studies have shown that infection by the virus in infancy is associated with a higher risk of developing subsequent bronchial obstructive disease. Much debate surrounds the mechanisms underlying this association. It is thought that a combined immunological and neurogenic response mechanism is likely. Prevention of severe respiratory disease in infants and young children with congenital heart disease due to infection by the virus may, therefore, offer both immediate and long-term benefits. Indeed, an increasing body of evidence supports this hypothesis, indicating a clinical rationale for prophylaxis against the virus in infancy, in order to reduce the chance of developing reactive airways disease and asthma in later life.

816. RNAi Applications for Respiratory Epithelia: Delivery and Efficacy

The airway epithelium is a common site of disease pathogenesis, including viral infections and primary diseases associated with inflammation. The delivery of RNAi to respiratory epithelia offers an approach to inhibit replication of RNA viruses and modify host responses to infection. To this end, we are using respiratory syncytial virus (RSV) as a model disease target. RSV is a major cause of respiratory disease in infants and immunocompromised adults. There is no approved vaccine for RSV, and medical treatment is supportive. The primary site of RSV replication is in the respiratory epithelium. As a proof of principle for RNAi delivery to the conducting airways of mice, we used gel-formulated adenoviral vectors to deliver short hairpin RNAs (shRNAs) against GFP or an irrelevant control to respiratory epithelia of GFP-transgenic mice. Vector transduced cells were visualized by expression of a reporter gene (dsRed or beta-galactosidase) to localize cells with putative RNAi activity. This approach achieved efficient transduction of the conducting airway epithelium. The efficiency of RNAi against GFP is measured by reduction of GFP in transduced cells. In a complementary approach, we are evaluating uptake of Cy3 labeled siRNA oligos in polarized human airway epithelial (HAE) cell culture and in mouse models. To inhibit RSV expression in vitro and in vivo, shRNAs were designed against the viral nucleocapsid (N) and large polymerase (L) genes of the RSV A2 strain and cloned into an expression vector behind a mU6 promoter. A misdirected control shRNA was produced by the same method to control for off-target effects of shRNA. Short hairpin expression vectors were screened for RNAi activity against renilla luciferase-viral gene fusions in co-transfection experiments, and functional constructs were identified. Subsequently, antiviral activity of effective shRNAs was measured in an in vitro infection model by reduction of viral RNA (Real Time-PCR) and viral protein (western blot). We will present our progress in these studies.

TARGETED THERAPY OF RESPIRATORY SYNCYTIAL VIRUS BY 2-5A ANTISENSE

Respiratory syncytial virus is a leading cause of respiratory disease in infants, young children, immunocompromized patients, and the elderly. Previous work has shown that RNase L, an antiviral enzyme of the interferon system, can be recruited to cleave RSV genomic RNA by attaching tetrameric 2′-5′-linked oligoadenylates (2 5A) to an antisense oligonucleotide complementary to repetitive intergenic sequences within the RSV genome (2 5A antisense). RBI034, a 2′-O-methyl RNA-modified analogue of the 2 5A anti-RSV compound, was found to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L· and sequence-specific manner. RBI034′s efficacy in suppressing RSV replication in cell culture is 50 to 100 times better than ribavirin, the only approved drug for RSV infection. Here we show that the activity of 2 5A antisense compound can be further enhanced by a combination treatment with interferon or ribavirin. The anti-RSV activity resulting from combination treatment is more potent than either treatment alone. We also demonstrate that RBI034 is effective against RSV in three different species: mice, cotton rats, and African green monkeys.