A bronchiolitis epidemic caused by respiratory syncytial viruses

In the typical period of the year (autumn-winter 1986-1987) an outbreak of bronchiolitis in infants caused by respiratory syncytial virus (RSV) occurred. The acute course of the illness, a severe clinical picture, and a very fast spread of infection called for the very rapid techniques for detecting the etiological agent. Applying the direct immunofluorescent antibody test (DFTA) in samples of nasopharyngeal secretions, RSV was found to be responsible for the epidemic. Later the same samples of the material were inoculated into tissue cultures and RSV was isolated. Fourfold or higher titres of complement fixing antibodies in paired serum samples confirmed the infection with RSV. During the outbreak of bronchiolitis 72 children aged under 15 months (only one child was older than one year) were hospitalized. The majority of cases of RSV bronchiolitis was noticed in infants in the first six months of life (81.9%, chi 2 = 31.1, p less than 0.01). Boys against girls were in relation as 1.25:1. Because of the broncho-obstructive component of the illness, respiratory failure was the dominant sign in the clinical picture of bronchiolitis; 34.7% of the patients were afebrile, 16.7% were in the state of hyperpyrexy and 48.6% with temperature up to 38 degrees C. Oxygen and symptomatic therapy were the basic treatment. In severe cases corticosteroids and antibiotic were administered for a short time. The classical type of bronchiolitis in infancy as the most severe among acute infections of respiratory organs because of its poor prognosis at the very beginning and the long-term sequels is a serious medical problem. Emphasis is laid on the importance and usefulness of the virologic diagnosis, especially the application of rapid DFTA techniques in the acute stages of bronchiolitis caused by infections with respiratory syncytial virus during the epidemic occurrence of this agent.     

Analysis of relatedness of subgroup A respiratory syncytial viruses isolated worldwide.

Respiratory syncytial virus strains (subgroup A) isolated from around the world during the period 1988-1991 were analysed to determine their relatedness. Analysis was by restriction mapping and nucleotide sequencing following amplification of selected regions of the virus genome by polymerase chain reaction (PCR). Twenty-three viruses of subgroup A isolated from cities in temperate regions of the Northern and Southern hemispheres and the tropics during the period 1988-1991 fell into distinct groupings closely related to four of the six lineages defined in analysis of recurrent epidemics within the same city (Birmingham, UK) during the same period. These observations confirm that multiple lineages of RS virus co-circulate locally, and show that very similar viruses are present simultaneously in widely separated countries.     

Expression of beta-galactosidase by recombinant respiratory syncytial viruses for microneutralization assay

The beta-galactosidase gene (lacZ) was inserted into a recombinant respiratory syncytial virus (RSV) A2 strain of subgroup A RSV (designated as A-lacZ) and a chimeric RSV that had the G and F surface glycoproteins of A2 replaced by those of the subgroup B RSV 9320 strain (designated as B-lacZ). Both recombinant RSVs, A-lacZ and B-lacZ, grew well in tissue culture and expressed high levels of beta-galactosidase. Using these two beta-galactosidase-expressing recombinant RSVs, a novel microneutralization assay was developed to measure serum anti-RSV neutralizing antibody from subgroup A or subgroup B RSV infection. The assay was carried out in 96-well plates and the unneutralized virus was quantitated by spectrophotometric measurement of the beta-galactosidase enzymatic reaction following incubation of the infected cell lysate with the enzyme substrate, chlorophenol red beta-D-galactopyranoside (CPRG). Adult human sera positive for anti-RSV antibody as shown by Western blot analysis and subgroup A or subgroup B RSV infected monkey sera were examined for the levels of anti-RSV neutralizing antibodies by the microneutralization assay in comparison with the plaque reduction neutralization assay. A higher antibody titer was detected when the neutralization assay was performed with the homologous RSV than the heterologous RSV, indicating that neutralization assay could distinguish antigenic differences between the two RSV subgroups. The microneutralization assay is comparable to the plaque reduction neutralization assay in sensitivity, but it is rapid, less laborious and suitable for screening a large number of samples.     

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.     

Concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co‐infections to the HRSV‐disease have been based on the detection of HRSV by RT‐PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co‐detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable‐HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT‐PCR and virus isolation in cell culture. All samples with viable‐HRSV were tested further by PCR for other respiratory viruses. HRSV‐disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV‐positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV‐positive diseases were more severe than HRSV‐negative ones, but there was no difference in disease severity between patients with viable‐HRSV and those HRSV‐positives by RT‐PCR. Co‐detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT‐PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co‐detection of other respiratory viruses was found in samples with viable‐HRSV, but this was not associated with more severe HRSV infection. J Med. Virol. 85:1852–1859, 2013. © 2013 Wiley Periodicals, Inc.

     

Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: a conserved feature among human and bovine respiratory syncytial viruses.

We investigated the nature of the oligosaccharide modification of the glycosylated forms of the small hydrophobic integral membrane protein, SH (previously designated 1A), of respiratory syncytial (RS) virus. Analysis of SH protein expressed in cells infected with RS virus or with a recombinant vaccinia virus revealed two glycosylated SH protein species, SHg and SHp, which contained N-linked carbohydrate residues. SHp migrated diffusely on polyacrylamide gels, which suggested modification by polylactosaminoglycan oligosaccharides. Polylactosaminoglycan modification of SHp was established from three lines of investigation: (1) the synthesis of SHp in a cell line (IdID) conditionally defective in the ability to add specific carbohydrate residues to N- or O-linked oligosaccharide chains required the addition of galactose, which is a component of the N-acetyllactosamine repeating unit; (2) SHp was sensitive to digestion with endo-beta-galactosidase, which cleaves the beta 1-4 linkage between galactose and N-acetylglucosamine of the repeated N-acetyllactosamine subunit; and (3) SHp was selected by Datura stramonium lectin (Dsl), which has specificity for polylactosaminoglycans. The presence of SHp as a component of purified human subgroups A and B and bovine RS virus particles was demonstrated by Dsl affinity selection. In addition to SHp, nonglycosylated SHo was selected by Dsl affinity, indicating that SHp and SHo may associate to form complexes within infected cells and virus particles. To identify conserved amino acid residues among the human and bovine SH glycoproteins that may function as signals for polylactosaminoglycan modification, the nucleotide sequences of the SH protein genes of a human subgroup B virus (8/60) and a bovine virus (391-2) were determined and compared to those of a human subgroup A virus (A2), a subgroup B virus (18537), and a bovine virus (A51908). A comparison of the deduced amino acid sequences of the human and bovine RS virus SH proteins indicated that a central hydrophobic region and the presence of potential N-linked glycosylation sites on either side of the central hydrophobic region were conserved features that may be required for the polylactosaminoglycan modification of SH.     

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.     

Interferon production by human mononuclear leukocytes: differences between respiratory syncytial virus and influenza viruses.

The ability of respiratory syncytial virus (RSV) to induce interferon production by human mononuclear leukocytes was compared with that of influenza viruses. Cell culture fluids were assayed for interferon activity 1, 3 and 7 days after exposure to RSV or to one of two subtypes of influenza A virus (H0N1 and H3N2). RSV induced interferon production inconsistently and in low titers. Varying the multiplicity of infection did not improve the ability of RSV to induce interferon production. In contrast, influenza viruses were effective inducers of interferon production. Seropositivity to the influenza virus strains was not associated with increased interferon titers. Interferon produced after exposure to RSV or to the influenza viruses was resistant to low pH treatment. The data suggest that interferon production may not be a major component of human immunological defense against RSV infection.     

Effect of 2-deoxy-D-glucose and glucosamine on the growth and functions of respiratory syncytial and parainfluenza 3 viruses.

Equilibrium properties and kinetics of the disassembly of coliphage fd induced by guanidine hydrochloride and sodium dodecyl sulphate were investigated by means of uv absorption, circular dichroism, and sedimentation. In both cases the phase disassembled in an all-or-none fashion, and the concentrations of the midtransition were 6.4 M and 0.03%, respectively, at 25°. The dissociation of the minor coat protein and the loss of infectivity were ascertained to take place in the disassembly. The DNA and major coat protein molecules released in guanidine hydrochloride solutions were both in nearly random coil conformations. In the sodium dodecyl sulphate disassembly the dissociated DNA molecule exhibited the same uv absorption as in the intact phage, and the dissociated major coat protein molecule remained in an approximately 50% α-helical conformation.     

Clustered charge-to-alanine mutagenesis of human respiratory syncytial virus L polymerase generates temperature-sensitive viruses

Clustered charge-to-alanine mutagenesis was performed on the large (L) polymerase protein of human respiratory syncytial virus to identify charged residues in the L protein that are important for viral RNA synthesis and to generate temperature-sensitive viruses. Clusters of three, four, and five charged residues throughout the entire L protein were substituted with alanines. A minigenome replicon assay was used to determine the functions of the mutant L proteins and to identify mutations that caused temperature sensitivity by comparing the level of reporter gene expression at 39 and 33 degrees C. Charge-to-alanine mutations were introduced into an antigenomic cDNA derived from RSV A2 strain to recover infectious viruses. Of the 27 charge-to-alanine mutations, 17 recombinant viruses (63%) were obtained. Seven mutants (41%) exhibited small plaque morphologies and/or temperature-sensitive growth in tissue culture. To generate mutant viruses with more temperature-sensitive and attenuated phenotypes, several clusters of charge-to-alanine substitutions were combined. Five combination mutants were recovered that exhibited shut-off temperatures ranging from 36 to 39 degrees C in tissue culture and restricted replication in the respiratory tracts of cotton rats.     

An investigation of media for the long term storage of three respiratory viruses

Summary A new transport medium based on agar and charcoal (AC) was compared with a recently modified conventional medium consisting of Hanks' balanced salt solution containing albumen and HEPES buffer (HH). Rhinovirus 2, influenza B and Coxsackievirus A21 were stored in the two media at room temperature, 4°, –20° and –70° C and titrated at various times. Rhinovirus 2 was stable for up to 17 weeks when stored at –70°, –20° or 4° C in HH medium or at –70° or 4° C in AC medium but was unstable in AC medium at –20° C. Influenza B survived well only at –70° C in HH medium and appeared to be inactivated by AC medium. Coxsackievirus A21 was stable in both media at –70° and –20° C. Collection of clinical material into both media suggested HH medium was superior for rhino-viruses. This medium was used in the Antarctic for the collection and transport of nasal swabs back to the U.K. Rhinovirus 2 was isolated from 79 of 268 swabs when they were examined over ten months later. This is the first time rhino viruses have been isolated from men in Antarctica and confirms the value of HH medium.W.H.O. Visiting Worker from Laboratoire National de la Santé Publique, 25 Boulevard Saint-Jacques, Paris, XIV, France.     

呼吸道合胞病毒分离株N蛋白编码基因特征分析

目的 阐明人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)A和B亚型病毒分离株的核蛋白(nucleoprotein,N)编码基因特征.方法 采用逆转录-聚合酶链反应(revelrse transciptionpolymerase chain reaction,RT-PCR)对北京2004年分离的HRSV代表株(2株A亚型和2株B亚型)进行N基因全长序列扩增、测序,并和GenBank下载的所有HRSV病毒的N基因全长序列进行对比和分析.结果 2株HRSV A亚型分离株与A亚型Long株(标准株)的N蛋白的核苷酸和氨基酸差异分别为36～40个(3.1%～3.4%)和4个(1.0%);2株HRSV B亚型分离株与B亚型CH18537株(标准株)的N蛋白之间的核苷酸和氨基酸差异分别为17(1.4%)和1(0.3%)个.4株HRSV代表株和从GenBank下载的HRSV的N蛋白之间的核苷酸和氨基酸差异分别为3～172个(0.25%～14.63%)和0-18个(0～4.6%).结论 N基因在HRSV基因组中是较为保守的基因,A或B亚型的型内差异相对较小;但A和B亚型之间N基因序列有较大变异,变异平均分配于整个N基因中;本研究为HRSV基因快速诊断试剂的研制提供了基因信息学数据.     

Nosocomial respiratory syncytial virus infections.

We studied the frequency and severity of respiratory syncytial virus infections acquired nosocomially on an infants' ward during a community outbreak. Every three or four days all infants and staff were examined, and specimens were obtained for viral isolation. During two months, 14 of 44 contact infants acquired the virus. All were ill, and four had pneumonia. Infected infants had a significantly longer mean hospital stay (21.5 days) than uninfected ones (9.2 days, P less than 0.001). Risk of nosocomial infection could not be related to age or to underlying disease, but was linked to length of hospitalization: 45 per cent of infants hospitalized for one week or more became infected, and the percentage increased with length of stay. Ten of 24 staff members also acquired the virus and appeared to play a major role as virus carriers. Nosocomial respiratory syncytial virus infection poses a major risk for hospitalized infants and adds to hospital costs.     

A comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus

Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group. Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and. bovine RS viruses have similar polypeptide profiles. However, PVM does not resemble the other two viruses closely. The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide. Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain. The mobilities of the other viral polypeptides showed little variation. The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin.     

Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients with respiratory symptoms

Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-time PCR increased the diagnostic yields for these viruses from 24% to 43% and from 3.5% to 36% for children and adults, respectively.     

Molecular characterization of respiratory syncytial viruses infecting children reported to have received palivizumab immunoprophylaxis

BackgroundRespiratory syncytial virus (RSV) is a major cause of respiratory infections in children. Palivizumab (PZ) is the only RSV-specific immunoprophylaxis approved by the U.S. Food and Drug Administration. Mutations leading to amino acid substitutions in the PZ binding site of the RSV F protein have been associated with breakthrough RSV infections in patients receiving PZ.ObjectiveTo detect PZ resistance conferring mutations in RSV strains from children who received PZ.Study designChildren aged ≤24 months on October 31 who were hospitalized or had outpatient visits for respiratory illness and/or fever during October–May 2001–2008 in 3 US counties were included. PZ receipt was obtained from parent interviews and medical records among children subsequently infected with RSV. Archived nasal/throat swab specimens were tested for RSV by real-time RT-PCR. The coding region of the PZ binding site of the RSV F protein was sequenced using both Sanger and pyrosequencing methods.ResultsOf 8762 enrolled children, 375 (4.3%) were tested for RSV and had a history of PZ receipt, of which 56 (14.9%) were RSV-positive and 45 of these had available archived specimens. Molecular typing identified 42 partial F gene sequences in specimens from 39 children: 19 single RSV subgroup A, 17 subgroup B and 3 mixed infections. Nucleotide substitutions were identified in 12/42 (28.6%) RSV strains. PZ resistance mutations were identified in 4 (10.2%) of the 39 children, of which one had documented PZ receipt.ConclusionsAlthough RSV PZ resistance mutations were infrequent, most RSV-associated illnesses in children with a history of PZ receipt were not due to strain resistance.     

Evaluation of five methods for respiratory syncytial virus detection.

A total of 117 nasal aspirates were cultured for respiratory syncytial virus (RSV) and tested for RSV antigen by a direct fluorescent-antibody (DFA) test (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), the Directigen enzyme immunoassay (EIA; Becton Dickinson Microbiology Systems, Cockeysville, Md.), the TestPack EIA (Abbott Laboratories, North Chicago, Ill.), and RSV EIA (Abbott). Agreement of two of five methods or a positive RSV culture were required to validate a result. A total of 57 of 117 (48.7%) specimens were culture positive in HEp-2 cells, A549 cells, or both. A total of 5 of 117 (4.3%) additional specimens met the criteria of a positive specimen; i.e., 62 of 117 (53.0%) specimens were positive. Results obtained from 77 of 117 (65.8%) specimens were concordant for all five methods. The sensitivities, specificities, and positive and negative predictive values for the culture and DFA methods were 91.9, 100, 100, and 91.7% and 91.9, 96.4, 96.6, and 91.4%, respectively. The sensitivities, specificities, and positive and negative predictive values for the three EIA procedures, Directigen, TestPack, and RSV EIA, were 75.8, 80.0, 81.0, and 74.6%; 93.6, 100, 100, and 93.2%; and 71.0, 100, 100, and 75.3%, respectively. New self-contained EIA configurations and the DFA method offer attractive alternatives to the culture method. Technical simplicity, rapid turnaround time, performance, and cost must all be considered when selecting a system for RSV detection.     

Monitoring epidemic viral respiratory infections using one-step real-time triplex RT-PCR targeting influenza A and B viruses and respiratory syncytial virus

Rapid and specific diagnosis of influenza A/B and respiratory syncytial virus (RSV) viruses is needed for optimal management of patients with acute respiratory infections. In this study, a one‐step triplex real‐time RT‐PCR assay was developed for rapid diagnosis of influenza A/B and RSV infections to optimize diagnosis efficiency of acute respiratory infections. Cell‐culture supernatants and clinical samples were used to evaluate specificity and sensitivity of the assay. The assay was used routinely during two winter epidemics for testing respiratory specimens from 2,417 patients. The limit of detection in cell‐culture supernatant was 1–10 plaque forming units/input (influenza A/B) and 2 × 10^?2 50% tissue culture infectious dose/input (RSV). In clinical samples, the assay was as sensitive as commercial molecular assays for the detection of each influenza A/B and RSV (Flu‐A/B and RSV‐A/B r‐gene?) individually, and far more sensitive than antigen detection. During the winter 2008–2009, the assay identified 145 RSV, 42 influenza A, and one mixed RSV‐influenza A infections among 298 patients. The next winter, the assay was used in two independent hospital laboratory settings. 776 patients were tested in one hospital and 1,343 in the other, resulting in 184 and 501 RSV, 133 and 150 influenza A, and 1 and 11 mixed RSV‐influenza A infections, respectively, being detected. This new user‐friendly assay allows rapid (within hours), effective molecular diagnosis of single or mixed infections involving influenza A (including seasonal A H1N1 and H3N2, and A(H1N1) 2009), influenza B, and RSV(A/B). The assay is very valuable for managing patients during winter epidemics when influenza and respiratory syncytial viruses co‐circulate. J. Med. Virol. 83:695–701, 2011. © 2011 Wiley‐Liss, Inc.