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PubMed2004

Association study suggests opposite effects of polymorphisms within IL8 on bronchial asthma and respiratory syncytial virus bronchiolitis.

BACKGROUND: IL-8 is a strong inductor of inflammation. Accordingly, it plays a pivotal role in acute inflammatory responses during respiratory syncytial virus (RSV) infections and in chronic inflammatory diseases such as bronchial asthma and juvenile idiopathic arthritis. Recently, 2 studies have found association of the polymorphism -251A of IL8 with RSV bronchiolitis. Furthermore, epidemiologic studies have demonstrated an increased risk for the development of asthma after RSV bronchiolitis, and a common genetic background for the 2 diseases is currently being discussed. OBJECTIVE: This study investigated whether IL-8 is in association with asthma and/or arthritis and whether the results can confirm a common genetic background of RSV bronchiolitis and asthma. METHODS: The polymorphisms -A251T, C781T, C1633T, and A2767T within IL8 were genotyped in the following 4 populations: children with asthma, atopic children, children with juvenile idiopathic arthritis, and control subjects. Statistical analysis made use of the Armitage trend test and the software program Arlequine. RESULTS: Association of all polymorphisms was found with asthma ( P =.008 to P =.03). Surprisingly -251T was associated with asthma, which is the opposite allele as described in association with RSV bronchiolitis. Furthermore, all polymorphisms were significantly more common in children with arthritis than in asthmatic children ( P =.006 to P =.02). No association was seen with the diagnosis of arthritis per se or with atopy. CONCLUSION: This is the first study to describe association of IL-8 with asthma and a significant inverse distribution of the polymorphisms in juvenile idiopathic arthritis. In addition, the results of this study might suggest that RSV bronchiolitis and bronchial asthma have at least some different genetic factors.

Adolescent↗
PubMed2005

Respiratory syncytial virus infection in the absence of STAT 1 results in airway dysfunction, airway mucus, and augmented IL-17 levels.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading infectious cause of respiratory failure and wheezing in infants and young children. Prematurity is the greatest risk factor for severe RSV-induced disease, and recent studies suggest that premature children have lower levels of the type I IFNs (alpha/beta), for which signal transducer and activator of transcription (STAT) 1 is a critical intracellular signaling molecule. OBJECTIVE: We hypothesized that RSV infection in STAT 1 knockout (STAT 1 KO) mice would result in both increased airway resistance and airway hyperresponsiveness. METHODS: Wild-type (WT) and STAT 1 KO mice on a BALB/c background were either RSV or mock infected. Phenotypic response to infection was assessed by means of plethysmography, immunohistochemistry, and lung cytokine measurement. RESULTS: We found that STAT 1 KO mice infected with RSV (STAT 1 KO-RSV) had greater baseline lung resistance (P=.05) and airway responsiveness (P<.001) than mock-infected STAT 1 KO (STAT 1 KO-MOCK), RSV-infected wild type (WT-RSV), and mock-infected wild type (WT-MOCK) mice. In addition, the STAT 1 KO-RSV mice showed induction of mucus production and expression of gob-5 and Muc5ac, conditions not present in any of the other 3 groups. IL-17, a cytokine that regulates Muc5ac expression, was expressed in the lungs of the STAT 1 KO-RSV mice, whereas lung levels of IL-17 were undetectable in the remaining groups. Expression of the IL-23-specific p19 subunit was also increased in the STAT 1 KO-RSV mice but not in the WT-RSV mice. CONCLUSION: These results show that STAT 1 has an important regulatory role in RSV-induced alteration of airway function.

Animals↗
PubMed2005

Respiratory syncytial virus and other respiratory viruses during the first 3 months of life promote a local TH2-like response.

BACKGROUND: Respiratory syncytial virus (RSV) infections during infancy are considered to be a risk factor for developing asthma and possibly allergic sensitization. OBJECTIVE: The aim of this study was to investigate the cytokines, chemokines, and eosinophil cationic protein in the nasopharyngeal secretions of infants < or = 7 months of age with RSV infections or other respiratory viral infections and healthy infants as controls. Groups were also analyzed according to age, < or = 3 months and >3 months, and the levels were compared within and between groups. RESULTS: Thirty-nine infants with RSV, 9 with influenza or parainfluenza virus infections and 50 controls with no history of infections, were enrolled in the study. The RSV-infected infants had significantly higher levels of IL-4; macrophage inflammatory protein 1beta, a chemoattractant for T cells; and eosinophil cationic protein in nasopharyngeal secretions compared with the control group. The levels of the TH2 cytokine IL-4 were significantly higher in RSV-infected infants < or = months of age compared with RSV-infected infants >3 months of age. In infants < or = 3 months of age, infections with influenza or parainfluenza virus caused TH2-like responses similar to those produced by RSV. CONCLUSION: Infections with RSV as well as with influenza and parainfluenza virus during early infancy preferentially promote a TH2-like response in the nose with local production of IL-4, IL-5, and macrophage inflammatory protein 1beta and infiltration and activation of eosinophils.

Age Factors↗
PubMed2004

Measurement of antibody against contemporary virus lineages of human respiratory syncytial virus sub-group A in infants and their mothers.

BACKGROUND: Human respiratory syncytial virus (hRSV) infects the majority of infants in their first year of life. Maternal antibodies offer some protection although a small proportion of infected infants develop bronchiolitis and require admission to hospital. A number of lineages of the virus co-circulate in the population and the prevalent virus lineage changes from epidemic to epidemic. The effect of antigenic variation between virus lineages upon the protection offered by maternal antibodies has not been assessed. OBJECTIVES: To explore the possibility that infants may develop bronchiolitis because of a virus lineage-specific deficiency in their maternal antibodies. STUDY DESIGN: Virus isolates from infants admitted to hospital in Newcastle upon Tyne with hRSV infection during two consecutive winter epidemics were classified into lineages by genotypic analysis. Antibodies to the surface glycoproteins of contemporary sub-group A lineages and to the A2 virus strain were assayed in the acute sera of infected infants, in a group of uninfected infants and in the mothers of both groups. RESULTS: Four lineages of sub-group A hRSV were found circulating during the study period. Antibody titres measured against all virus lineages in the acute serum of infants with hRSV bronchiolitis were similar. In the uninfected infants and in the mothers of both infected and uninfected groups antibody titres to all four contemporary virus lineages were also similar. However, in these groups antibodies to the A2 virus strain were four-fold lower than those to contemporary isolates. CONCLUSIONS: Infants admitted to hospital with hRSV bronchiolitis exhibited no apparent selective deficiency in maternal antibodies to the viral glycoproteins of the infecting virus strain or lineage.

Antibodies, Viral↗
PubMed2004

Comparison of two new rapid antigen detection assays for respiratory syncytial virus with another assay and shell vial culture.

BACKGROUND: Respiratory syncytial virus (RSV) is the major viral pathogen causing lower respiratory tract infection in young children. Early detection allows cohorting of infected inpatients to prevent nosocomial transmission and consideration of treatment. To achieve rapid reporting, antigen detection has been widely used. Since late 2002, the FDA approved two new RSV antigen detection tests, the Now RSV test (Binax) and the Directigen EZ RSV (Becton Dickinson), both of which promised reduced hands-on processing. OBJECTIVES: Evaluate relative performance of the two new rapid antigen detection assays. STUDY DESIGN: To compare the performance of these newer tests with a previously existing antigen detection test (Becton Dickinson) and shell vial viral culture with clinical specimens. RESULTS AND CONCLUSIONS: A total of 89 fresh respiratory specimens from 84 pediatric patients were tested. We also performed reverse transcription-polymerase chain reaction (RT-PCR) on all culture negative but antigen positive samples as well as 26 specimens that were negative on both shell vial culture and antigen testings. Overall, the three antigen assays performed similarly well. The hands-on processing for the two newer tests was significantly reduced, making them more convenient for rapid screening.

Adolescent↗
PubMed2004

Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients. OBJECTIVES: To rapidly and accurately quantify and subtype RSV in respiratory samples, we developed and evaluated two real-time RT-PCR assays. STUDY DESIGN: A quantitative assay was designed using primers for a consensus region of the matrix protein gene and a subtype-specific assay for RSV-A and RSV-B detection was designed using primers for the polymerase gene. Quantitative RSV RT-PCR results of pediatric nasal wash samples submitted to the University of Washington Virology Laboratory from December 2002, through May 2003, were compared to those of an indirect fluorescent antibody RSV antigen detection assay (FA). RESULTS: Specificity of the RT-PCR assay was high, with no amplification of eleven common respiratory viruses and eight herpes viruses. Among 751 samples, RSV was detected in 267 (35.6%) by FA and in 286 (38.1%) by RT-PCR. Median RSV copy number in nasal wash samples that were positive by both FA and RT-PCR was 2.5 x 10(7) copies/mL versus a median of 3.0 x 10(4) copies/mL for samples positive by RT-PCR only (P < 0.001). The detection and quantity of RSV in respiratory specimens was associated with younger age, but not with gender or hospitalization. Among positive samples from this Seattle cohort, 52% were subtype A and 48% were subtype B. Both subtypes were detected with similar viral loads among all patient groups (stratified by age, gender, and hospitalization), and throughout the specimen collection period. CONCLUSIONS: These real-time RT-PCR assays provide a rapid, specific, and highly sensitive alternative for detecting, quantifying, and subtyping RSV in clinical specimens.

Adolescent↗
PubMed2005

A comparison of Thermo Electron RSV OIA to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

BACKGROUND: Rapid diagnostic methods for respiratory syncytial virus are useful tools available for the clinician. OBJECTIVES: The Thermo Electron RSV OIA (optical immunoassay kit) was prospectively compared with direct immunofluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah. STUDY DESIGN: Specimens from three hundred and thirty patients exhibiting respiratory symptoms were collected for testing by the three methods above. Several specimens were positive by both OIA and DFA with a negative culture result. These culture results were verified by RT-PCR analysis. RESULTS: Overall, 107 specimens were positive for RSV by the reference tests (culture or RT-PCR). DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests the sensitivity, specificity, positive, and negative predictive values of the OIA for detection of RSV were 87.9%, 99.6%, 98.9% and 94.5%, respectively. CONCLUSIONS: The rapid OIA assay format proved to be cost effective, and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.

Adolescent↗
PubMed2005

Genetic characterization of respiratory syncytial viruses isolated from consecutive acute respiratory infections in a HIV infected child.

BACKGROUND: Human respiratory syncytial virus (HRSV) was isolated from a 2-year-old child suffering from perinatally transmitted AIDS in the course of three distinct episodes of respiratory infections. The first episode occurred in the winter of 1994, the following two episodes of cough and fever occurred two and 4 months after the initial episode. OBJECTIVE: To analyze the genetic variability of the child's HRSV strains, and with contemporary circulating HRSV isolates. RESULTS: The three child's HRSV isolates belonged to group B. Sequence analysis of the attachment (G) protein gene (which has the highest degree of antigenic and genetic diversity in HRSV), demonstrated no difference in the sequence obtained from the three isolates recovered from the child. Comparison of the child's HRSV strain with contemporary circulating group B HRSV isolates showed a close sequence similarity with one of them. CONCLUSIONS: The immunodeficiency in an HIV-positive child may have resulted in the recurrent isolation of one HRSV strain. Although it cannot be discarded the possibility that the recurrent episodes might be re-infections, it is unlikely in view of the lack of change in the HRSV glycoprotein G. This is the first study that analyzes the genetic variation in HRSV isolates from consecutive respiratory disease episodes in an immunosuppressed patient.

Acute Disease↗
PubMed2006

Detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness.

BACKGROUND: Reverse transcription polymerase chain reaction (RT-PCR) is a powerful tool that allows the detection of minute quantities of viral RNA. Because of the sensitivity of these assays it is possible that the finding of viral RNA indicates not only active infection but also transient colonization or residual nucleic acid from a distant infection. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two viruses for which RT-PCR is now frequently used for diagnosis in adult disease. OBJECTIVE: We evaluated nasal secretions from adults with and without respiratory illnesses by nested, one-tube RT-PCR for RSV and hMPV to determine if rates of detectable RNA were significantly higher among ill subjects compared to controls suggesting a causal relationship with respiratory illness. METHODS: Adults presenting to a health care provider with complaints of respiratory illness were recruited as "cases" and those visiting for non-respiratory complaints were recruited as "controls". Subjects were enrolled during a 3-month period (January to April) when both viruses were expected to be prevalent in the community. Nasal swab samples were obtained and subjected to one-tube nested RT-PCR for RSV and hMPV. RESULTS: Of 146 ill subjects, 17 (11.6%) tested positive for RSV and 5 (3.4%) were positive for hMPV. Of the 158 control subjects, one was RT-PCR positive for RSV and none tested positive for hMPV. The rates of RT-PCR positive cases compared to controls were significantly different for RSV (p<.0001) and hMPV (p<.02). Subjects remained RSV RT-PCR positive on average until day 7.1+/-2.8 of symptoms with a range of 3-10 days. No subject had a positive swab on days 14, 21 or 28. CONCLUSION: Asymptomatic carriage of RSV or hMPV is uncommon. RT-PCR should be a useful method for the diagnosis of these viral illnesses in adults.

Adult↗
PubMed2006

Five-year cohort study of hospitalization for respiratory syncytial virus associated lower respiratory tract infection in African children.

OBJECTIVES: To describe the epidemiology of respiratory syncytial virus (RSV) associated lower respiratory tract infection (RSV-LRTI) hospitalizations in South African children over a 5-year period, and determine the impact of gestational age (GA) on the incidence of RSV-LRTI hospitalization. STUDY DESIGN: A cohort of 39,836 children, 6.47% of whom were HIV infected, enrolled into a phase 3 trial were prospectively studied for respiratory viruses when hospitalized for LRTI. RESULTS: The incidence of hospitalization for RSV-LRTI was 19.4 per 1000 in HIV uninfected children and 2.5-fold (95% CI 2.04-3.03) greater in HIV infected children (45.0 per 1000). The incidence of RSV-LRTI was 4.9-fold greater (95% CI 3.9-6.8) in children born at <36 weeks of gestational age (GA) and repeat hospitalizations for RSV-LRTI was 3.7-fold (95% CI 1.4-9.4) more likely in these children (7.3%) than children born at > or =36 weeks of GA (1.9%). The burden of RSV-LRTI was greater in children born at <32 weeks of GA than those born at 32-35 weeks of GA between 6-12 months (P=0.008) and 12-24 months of age (P=0.001). The RSV epidemic occurred at the end of the rainy season and peaked when the monthly temperatures were at its lowest each year.

Cohort Studies↗
PubMed2006

Novel mutations in the respiratory syncytial virus G gene identified in viral isolates from a girl with severe combined immune deficiency treated with intravenous immune globulin.

BACKGROUND: Respiratory syncytial virus (RSV) can cause prolonged infections in individuals with compromised immunity. OBJECTIVES: To investigate whether RSV evolves during prolonged infection in an immunocompromised host. STUDY DESIGN: We sequenced the envalope glycoprotein genes of RSV obtained at three time points during a 59-day period from a 4-year-old female with severe combined immune deficiency (SCID) treated with intravenous immunoglobulin (IVIG). RESULTS: Sporadic silent mutations were found in the SH, G and F genes among three RSV samples collected at days 0, 19 and 59. Premature stop codons at amino acid positions 257 and 278 were present in the RSV G glycoprotein gene sequenced from each time point. None of the 48 RSV G sequences available on GenBank or any of 50 genetically diverse clinical isolates of RSV contained these mutations. These premature stop codon mutations occurred at the same positions of the G glycoprotein gene as those described in in vitro monoclonal-antibody resistant mutants reported elsewhere. CONCLUSION: Our patient was most likely infected with a single RSV strain that did not mutate during the study period. This strain contains unique mutations that may have previously evolved in this individual who had prolonged infection and was treated with monthly IVIG.

Amino Acid Sequence↗
PubMed2005

Analysis of the binding of monoclonal and polyclonal antibodies to the glycoproteins of antigenic variants of human respiratory syncytial virus by surface plasmon resonance.

The surface glycoproteins of human respiratory syncytial virus (hRSV), F and G, are the major protective antigens of the virus. Both are antigenically variable, although to different degrees, but the role of antigenic variation in the pathogenesis of hRSV disease has not been fully evaluated. Assessment of immunity to different virus strains is difficult with conventional antibody assays where differing properties of the virus antigens, other than antigenicity, may influence the outcome of the assay. Here, we have developed BIAcore surface plasmon resonance based assays for antibodies to the glycoproteins of hRSV which allow valid comparison of antibody titres against multiple hRSV strains. Glycoproteins from a number of lineages of hRSV sub-group A were captured from lysates of infected cells onto the dextran coated surface of a BIAcore sensor chip via primary monoclonal antibodies (MAbs) to conserved epitopes. For the G glycoprotein, primary MAbs were conjugated directly to the dextran of the sensor chip via free amide groups. For the F glycoprotein, direct conjugation was found to inactivate the MAb and primary MAb was immobilised on the chip via rabbit anti-mouse Fc antibody fragments in an indirect system. Using monoclonal antibodies as secondary MAbs, the glycoproteins in both systems were shown to exhibit a sub-set of conserved and variable epitopes, with some epitopes of both sorts being unavailable, presumably blocked by the primary antibody. Polyclonal anti-hRSV sera raised against viruses of different genotype bound equally to both F and G glycoproteins from homologous and heterologous viruses suggesting that mice immunised systemically with lysates of cells infected with recent isolates of virus do not respond well to genotype specific epitopes.

Animals↗
PubMed2004

An unusual case of thrombocytosis associated with concurrent cytomegalovirus and respiratory syncytial virus infection in an immunocompetent infant: possible roles of thrombopoietin and interleukin-6.

This is the case study of concurrent cytomegalovirus and respiratory syncytial virus infection in an infant who showed thrombocytosis, liver dysfunction and bronchiolitis. The combination of thrombocytosis with this co-infection is causally related to elevated levels of thrombopoietin and interleukin-6. This study represents the first such case ever recorded.

Antibodies, Viral↗
PubMed2006

Impact of respiratory syncytial virus on hospital admissions in children younger than 3 years of age.

BACKGROUND: Respiratory syncytial virus (RSV) is the main pathogen associated to acute respiratory infections (ARI) in children worldwide. OBJECTIVE: To determine the contribution of RSV to hospital admissions in children <3 years of age from May 2003 through April 2005 in San Luis Potosí, Mexico. METHODS: Cross-sectional survey of children <3 years of age admitted to a public general hospital and detection of RSV in respiratory secretions of children with ARI. RESULTS: There were 2036 children <3 years of age admitted to the hospital during the study period. The diagnosis at the time of admission was an ARI in 734 (36.1%) patients. RSV was detected in 153 (24.8%) of the 616 subjects who were tested. Children <1 year comprised 66% of admissions among children <3 years. RSV was detected in 26.9% of children with ARI <1 year; 21.5% of those 12-24 months of age and 15.25% of those 24-36 months old. CONCLUSIONS: RSV was associated to 24.8% of ARI-related admissions in children <3 years of age. It is necessary to determine risk factors for RSV-related hospitalizations to evaluate the feasibility of establishing a preventive program for RSV infections in Mexico.

Acute Disease↗
PubMed2006

Respiratory syncytial virus inhibits interferon-alpha-inducible signaling in macrophage-like U937 cells.

Monocytes become susceptible to respiratory syncytial virus (RSV) infection when pretreated with phorbol 12-myristate 13-acetate (PMA). The molecular mechanism underlying this observation is poorly understood, but may be related to inhibition of type I interferon (IFN) signaling by RSV in epithelial cells. Herein, we have investigated the putative role of suppressor of cytokine signaling (SOCS) in the IFN-inducible antiviral response in U937 cells. Upon RSV infection of macrophage-like U937 cells, the expression of SOCS1, SOCS3, and CIS mRNA was rapidly upregulated, and phosphorylation of the IFN-alpha-inducible signal transducer and activator of transcription (STAT1 and STAT2) was suppressed. These results suggest that RSV can inhibit the phosphorylation of IFN-alpha-inducible STAT1 and STAT2 by inducing the expression of SOCS proteins in PMA-treated U937 cells.

Humans↗
PubMed2005

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization.

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay. Our data reveal a major differential effect of the association and dissociation rates on the RSV neutralization, particularly for intact antibodies wherein the association rate plays the predominant role. Furthermore, we found that antibody binding valence also plays a critical role in mediating the viral neutralization through a mechanism that is likely unrelated to antibody size or binding avidity. We applied an iterative mutagenesis approach, and thereafter were able to identify palivizumab Fab variants with up to 1500-fold improvement and palivizumab IgG variants with up to 44-fold improvement in the ability to neutralize RSV. These anti-RSV antibodies likely will offer great clinical potential for RSV immunoprophylaxis. In addition, our findings provide insights into engineering potent antibody therapeutics for other disease targets.

Amino Acid Sequence↗
PubMed2004

Development of a highly sensitive semi-quantitative real-time PCR and molecular beacon probe assay for the detection of respiratory syncytial virus.

Molecular beacons are a novel class of oligonucleotide probe capable of reporting the accumulation of target amplicon during real-time PCR by the emission of a fluorescent signal. A novel assay for the detection and estimation of respiratory syncytial virus (RSV) nucleic acid in clinical specimens based on real-time PCR utilising such a probe was developed. The probe consisted of two short arm sequences and a central loop sequence complementary to a region of the N gene (the target amplicon). The probe was characterised and a semi-quantitative nested real-time PCR using a LightCycler instrument was optimised. Standard curves were generated using cycle threshold (C(t)) values calculated from several assays over a range of logarithmic RSV titres. Linear coefficient correlations were close to one ( r(2) = 0.998) and the detection limit of the optimised assay was reproducibly shown to be 1 x 10 (-4) pfu/ml. The intra-assay coefficient of variation (CV) of C(t) values of the optimal assay was 0.8% and the CV of quantification data was 6.6%. The interassay CV of C(t) values was 2.0% and the quantification CV was 6.7%. The validity of the assay for the detection of RSV in clinical specimens was assessed by analysing ten specimens previously assayed in a different laboratory. Real-time PCR analysis was completely consistent with the results of prior analysis. The rapidity, sensitivity and specificity of the assay should greatly facilitate epidemiological studies, particularly in adults as existing methods perform better on clinical specimens from children.

Humans↗
PubMed2004

Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a ubiquitous RNA virus of the family Paramyxoviridae that may interfere with graft tolerance and with other interstitial lung diseases. The low viral titre observed in the immunodeficient transplanted patients requires a highly sensitive detection method. Although different tests already exist for the detection of RSV, reverse transcription-polymerase chain reaction (RT-PCR) has been shown to have the best sensitivity. In this study, a SYBR Green assay was established for the detection of RSV A and RSV B in a common screening test, and two quantitative TaqMan RT-PCRs were developed to quantify both RSV subgroups separately. Standard dilutions obtained from RSV cell infections were included in each test, and the assay was normalised using a housekeeping gene. RSV was found in 16% of the transplanted patients tested. The quantitative TaqMan assay is fast, reproducible, specific and very sensitive, and could facilitate considerably the detection of RSV virus. This would in-turn facilitate studies on the role of RSV in graft rejection.

Benzothiazoles↗