Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus

In order to study variation among prototype strains and clinical isolates of respiratory syncytial (RS) virus, four prototype strains (Long, A2, CH18537, 9320) were used to produce monoclonal antibodies to this virus. The majority of monoclonals reacted with all four prototype strains by fluorescent antibody staining. Among the non-cross-reacting monoclonals, five additional patterns of reactivity with the prototype strains were recognized. Fourteen monoclonals, including ones representative of each of the patterns of reactivity with the prototype strains, were selected to use for typing prototype strains and community isolates. All 14 were found by immunoprecipitation to recognize the RS virus G glycoprotein. These monoclonals could uniquely identify each of the prototype strains. In addition to the antigenic differences among the prototype strains detected by the monoclonals, differences were also detected in the migration of the G glycoprotein of the prototype strains in polyacrylamide gel electrophoresis. Fluorescent antibody staining with panels of monoclonals distinguished two antigenic types among 114 isolates of RS virus recovered from children in St. Louis during the period 1981-86. The predominant type (80% of isolates) had a pattern of reactivity that resembled but differed from that of either the Long or A2 strains. The second type had a pattern of reactivity identical with that of 9320. The possible significance of this heterogeneity must be considered in developing diagnostic tests as well as active or passive immunotherapy for infections caused by RS virus.     

Prevalence of respiratory syncytial virus IgG antibodies in infants living in a rural area of Mozambique

A case control study was carried out in Manhiça (Mozambique). Serum samples were collected from infants < 1 year of age in hospital to assess the effect of serum antibodies on the incidence of respiratory syncytial virus (RSV) infection. Sera were collected from a total of 31 cases of RSV infection and paired uninfected controls matched for age and sex. Anti‐RSV antibodies were assessed by a membrane fluorescent antibody test (MFAT) for immunoglobulin G (IgG) antibodies and by a neutralizing antibody test. IgG RSV antibodies were of higher prevalence and at higher levels in the control group when compared to the infected case group (P < 0.001), indicating an important role for IgG antibodies in protection. To assess infection before recruitment, IgA RSV antibodies were also measured by MFAT. IgA RSV antibody prevalence was very low in patients and controls (0/31 and 4/31 respectively), suggesting that most of the detected IgG RSV antibody in both groups was of maternal origin. Re‐analysis of data from the subset of 27 matched, IgA RSV antibody negative infant pairs mirrored the full analysis indicating that maternal antibody has an important role in RSV protection. Similar results were obtained when neutralizing antibodies were measured and when the measurement was done against subgroup A virus strain A2, subgroup B virus strain 8/60 and a contemporary subgroup A isolate, Moz00. No significant differences in the reactivity of maternal antibodies with the three virus strains were observed. The data described below represent the first analysis of the role of maternal antibodies in reducing the risk of pediatric infection in developing countries. The results reinforce the concept of maternal vaccination for the control of RSV in very young children in whom the risk and severity of infection are the highest. J. Med. Virol. 67:616–623, 2002. © 2002 Wiley‐Liss, Inc.     

Serotyping of herpes simplex virus isolates: A comparison of BVDU sensitivities,indirect immunofluorescence with monoclonal antibodies,and indirect immunofluorescence with cross-adsorbed rabbit antibodies

Four methods for typing of herpes simplex virus (HSV) isolates were compared for 43 recent clinical isolates and 3 reference strains of HSV, These isolates were subjected to indirect immunofluorescence using both monoclonal antibodies and cross-adsorbed rabbit antisera. Sensitivity to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was also examined.All isolates which fluoresced with HSV-1 monoclonals were found to be sensitive to BVDU with ID₅₀'s ranging from 0.001 to 0.006 μg/ml. All isolates labelled as HSV-2 using monoclonal antibodies had ID₅₀'s to BVDU ranging from 0.4 to 3.5 μg/ml. Results of typing with rabbit cross-adsorbed antisera were less accurate, however. When dilutions were predetermined according to manufacturer's instructions, only 3 of 22 isolates (14%) of HSV-1 were correctly typed. HSV-2 isolates were correctly labelled in 24 of 26 situations (92%).When fluorescence dilution endpoints were compared, however, 21 of 22(95%) HSV-1 isolates had fluorescence endpoints at a greater dilution with HSV-1 antiserum. Twenty-three of 24 HSV-2 isolates were also correctly typed (96%).     

Evaluation of clinical specimens for the presence of respiratory syncytial virus antigens using an enzyme immunoassay

An enzyme-linked immunoassay (EIA) was developed for the detection of respiratory syncytial virus (RSV) antigen in nasopharyngeal secretions. This assay, which employs goat and rabbit anti-RSV as the capture and detector antibodies respectively, was used in a retrospective evaluation of frozen clinical specimens from children. The EIA results were compared with those of virus isolation in cell culture and direct fluorescent antibody staining performed at the time of specimen collection. The sensitivity of the RSV EIA compared to cell culture was 91.3% (63/69) with a specificity of 96.8% (93/96). The predictive value of a positive EIA result was 95.4% and for a negative EIA result, 93.9%. The sensitivity of the RSV-EIA compared to direct FA was 91.5% (43/47) with a specificity of 96.5% (83/86). These data represent the preclinical evaluation of the Abbott RSV-EIA. This assay could prove to be a useful alternative to virus isolation or direct FA for the diagnosis of RSV infection.     

Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation

Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10–1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first postpartum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.     

静脉注射用丙种球蛋白对呼吸道合胞病毒治疗的作用

目的:研究静脉注射用丙种球蛋白(IVγG)体内外抗呼吸道合胞病毒(RSV)的作用。方法:①采用微量细胞病变抑制法试验(MCIA),检测不同浓度的IVγG抑制RSV的作用。②建立RSV感染的BALB/c小鼠模型,将40只感染RSV的小鼠分为4组,即IVγG治疗组、病毒唑治疗组、Palivizumab治疗组、生理盐水对照组,及10只未感染RSV的小鼠作为正常对照组,每组10只。于治疗后第5及第7天,分2批处死小鼠,分离病毒并观察肺组织的病理积分。结果:①IVγG具有抑制RSV的作用,其治疗指数(TI)为275,较病毒唑高6倍,较Palivizumab低2倍。②IVγG治疗组小鼠肺组织的病理积分较生理盐水对照组明显减低(P<0.01),但其5d的治疗作用低于Palivizumab治疗组(P<0.05)。结论:IVγG在体内外均有一定的抗RSV的作用,但效果均低于Palivizumab。     

呼吸道合胞病毒F蛋白研究进展

人呼吸道合胞病毒（RSV）融合蛋白（F蛋白）为病毒表面结构蛋白,可以刺激机体产生保护性抗体和细胞免疫反应。因此对F蛋白的深入研究将有助于了解病毒感染的机理,从而为研究RSV的免疫机理、研制特定部位的亚单位或多肽疫苗提供帮助。     

Diagnosis of respiratory syncytial virus infection in children: Comparison of viral antigen detection and serology

Direct detection of viral antigen in nasopharyngeal secretion by radioimmunoassay was compared with serology by IgG antibody enzyme immunoassay for diagnostic efficacy in 77 children with clinically suspected respiratory syncytial virus (RSV) infections. Antigen detection gave a positive diagnosis in 26 of 33 (79%) children in whom RSV infection was diagnosed by any of the two methods. The diagnostic efficacy of antigen detection was dependent upon the interval after onset at which specimens were collected; 88% of specimens taken during the first 5 days and 50% of specimens taken 6–10 days after onset of illness were positive. It was also dependent on the age of the patients, the diagnostic efficacy being 88 and 76% in children under and over 6 months of age, respectively.     

Role of complement in neutralization of respiratory syncytial virus

Serum neutralizing antibody titers to respiratory syncytial virus (RSV) are higher when assayed with guinea pig complement. A number of different mechanisms have been suggested for enhancement of neutralization by complement. The most straightforward is that complement-antibody complexes present a greater steric hindrance to viral entry than with antibody alone. To define the implications of measuring serum neutralizing antibody with and without complement, sera from adults, young children, infants, and cord bloods were run in plaque neutralization assays with representative viruses of the RSV A and B subgroups. Although titers of neutralizing antibody were higher in the presence of complement, the addition of complement did not increase the ability to detect antibody rises after natural infection. Some of the complement effect may be attributable to an inhibition of RSV replication by complement alone. While these observations do not address the role for complement in the pathogenesis of RSV infection, they suggest that neutralization assays performed without complement may be most reflective of physiologic conditions in the respiratory tract.     

Cellular reactivity to respiratory syncytial virus in human colostrum and breast milk

Colostrum and breast-milk samples were taken from 23 mothers between 2 days and 7 weeks postpartum and were examined for the presence of cellular reactivity to respiratory syncytial (RS) virus using a lymphocyte transformation assay. Positive responses were detected in nine of the 23 (39%) samples taken at 2-5 days postpartum, but this reactivity was undetectable at 3 weeks. Positive responses developed in a further three mothers during the 3-7-week period of lactation, suggesting a response to virus infection. Colostral whey was found to suppress the cellular response to RS virus and inhibition was related to the level of specific IgA antibody to RS virus present in the whey. The role of colostral cellular reactivity in protection of breast-fed infants from RS virus bronchiolitis is discussed.     

Maternal antibody and respiratory syncytial virus infection in infancy

One hundred newborn infants were studied prospectively for 1 year for evidence of infection with respiratory syncytial virus (RSV). The indirect membrane fluorescence technique was used to determine specific antibody in sera. Infection was shown in 29 cases. In 31 infants exposed to an RSV epidemic season, there was no evidence of infection. Maternal antenatal sera were also tested, and a wide range of IgG antibody to RSV was found. Mean titre of maternal IgG antibody to RSV was significantly higher (P < 0.001) in those mothers whose babies remained uninfected than in those whose babies had proved RSV infection before 6 months of age. Babies born to mothers with high levels of IgG antibody to respiratory syncytial virus were protected against infection with this virus during the first months of life when the risk of severe disease was greatest.     

Cell-mediated immunity in respiratory syncytial virus disease

Systemic cell-mediated responses to respiratory syncytial (RS) virus were detected, using a whole blood transformation assay, in 10 of 28 infants and children with RS virus infections during the period 1–14 days postadmission. Cell-mediated responses were unrelated to the age of the patient or the severity of illness. No correlation was found between cellular responses and fourfold or greater rises in antibody titre to RS virus, as determined by a membrane immunofluorescence technique. Patients under 6 months of age had significantly lower levels of IgA and IgG antibody to RS virus compared to older patients, although cell-mediated responses were similar in both groups. The presence of cell-mediated reactivity to RS virus was also demonstrated in 5 of 95 samples of cord blood examined, and cellular responses failed to correlate with the levels of IgG antibody to RS virus.     

Improvement of respiratory syncytial virus replication in actively growing HEp-2 cells

The growth of respiratory syncytial (RS) and parainfluenza type III (PI3) viruses has been studied in actively growing versus relatively stationary HEp-2 cells. There was no effect on PI3 virus growth. RS virus synthesis was stimulated from 20- to 60-fold by growth in actively growing cultures when the cell density was approximately $\text{1}\text{3}$ that of a confluent culture. This stimulation was manifested by a greater yield of virus per cell, more virus-specific mRNA produced per cell, and a 50% decrease in the time before cytopathic changes appeared.     

Dynamics of local defensive reactions in the lungs in acute experimental inflammation induced by respiratory syncytial virus

Laboratory of General Pathological Anatomy, Research Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 5, pp. 512–515, May, 1991.     

Experimental respiratory syncytial virus infection of four species of primates.

Four species of nonhuman primates were inoculated intranasally with 10(3.1) to 10(3.7) plaque forming units (pfu) of respiratory syncytial (RS) virus. Adults squirrel monkeys and newborn rhesus monkeys became infected and shed small quantities (peak titer 10(2.0) pfu/ml of nasopharyngeal swab specimen) of virus, but illness did not develop. Infant cebus monkeys aged 2 months became infected, shed 10(2.3) to 10(3.8) pfu/ml of nasopharyngeal swab specimen, but did not become ill. Chimpanzees aged 15 to 18 months shed a large quantity of virus, up to 10(6.0) pfu/ml of nasopharyngeal swab specimen and developed an upper respiratory illness. Chimpanzees are proposed as a possible animal model for future study of the immunopathology of RS virus disease and for in vivo evaluation of attenuated live virus vaccine candidates.     

Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya

Respiratory syncytial virus (RSV) causes significant burden of disease during infancy and childhood. This study examined the genetic relatedness of RSV positive samples from child inpatients and outpatients and a birth cohort from a rural coastal district of Kenya and also the distribution of strains between these three groups. Clinical samples were collected over a 4-year period in Kilifi District, Kenya from community and hospital surveillance. Three hundred ninety seven of 1,044 nasal specimens from children (under 5 years old) attending Kilifi District Hospital, and from community-monitored infants, were positive for RSV by multiplex RT-PCR. Of these, 376 samples were analysed further by restriction fragment length polymorphisms (RFLP) of the nucleocapsid (N) and attachment (G) protein genes. The G gene was sequenced for 109 samples and phylogenetic analysis carried out. The group A samples from Kilifi fell into two clusters based on G gene sequences, while only one group B cluster was observed. One RSV-B sample from 2003 demonstrated the presence of a 60-nucleotide duplication within the G gene, clustering with similar isolates from Buenos Aries from 1999. All had similar sequences to isolates from the UK, USA, Spain, or Uruguay. The Kilifi District samples showed greater than 97% homology to isolates from South Africa and Mozambique and 91-94% homology to isolates from The Gambia. Samples from different sources, clearly differing in disease severity, did not differ in genotype characteristics, suggesting that disease causing variants are a general reflection of infections within this community.     

Seroepidemiology of respiratory syncytial virus in western India with special reference to appropriate age for infant vaccination

Respiratory syncytial virus (RSV) causes significant infant mortality worldwide and a vaccine may be available soon. This study determined age-stratified anti-RSV antibody positivity (enzyme-linked immunosorbent assay [ELISA]) at Pune, India (cord blood-85 years). Antibody positivity declined from 100% at birth to 71.3% (3 months), and 0.7% (6 months). A significant rise was noted at 15 months (16%), 16 to 24 months (64.5%) and 4 years (95.2%) with concomitant IgM-anti-RSV positivity indicative of recent infection. Antibody decline was higher in infants born preterm than full-term. Across subsequent age groups including the elderly, antibody positivity was similar and comparable, suggestive of repeated exposure to the virus. Early protection/vaccination is essential for the infant population.     

Genetic predisposition to wheeze following respiratory syncytial virus bronchiolitis

BACKGROUND: The nature of the association between severe respiratory syncytial virus (RSV) bronchiolitis and subsequent wheezing remains unknown. In a previous study, we showed that genetic variation in the IL-8-promoter region is associated with susceptibility to severe bronchiolitis. OBJECTIVE: The purpose of this study was to assess the association between wheezing post-bronchiolitis and the genetic variant of IL-8 gene. METHODS: We collected data from 134 children who had suffered from bronchiolitis, enrolled in our previous study. The occurrence of wheezing post-bronchiolitis was recorded from a questionnaire sent by post. The association between the genotype and wheezing phenotype was assessed by family-based and case-control approaches. RESULTS: Family-based association showed that the IL-8 variant was transmitted significantly more often than expected in the children who wheezed after the episode of bronchiolitis (transmission=56%, P=0.02). This effect was not observed in the group of children who had bronchiolitis but did not go on to wheeze. Moreover, the variant was significantly more frequent in post-bronchiolitis wheezers compared with the general population (odds ratio=1.6, 95% confidence interval 1.0-2.6). CONCLUSION: These preliminary results suggest that there is a genetic predisposition to wheeze following severe RSV bronchiolitis.     

Occurrence and severity of infections caused by subgroup A and B respiratory syncytial virus in children in southeast Brazil

The frequency and severity of infections caused by respiratory syncytial virus (RSV) were assessed in children <2 years of age seen at the emergency department. The frequency of RSV detection in the clinical virology laboratory during the past 3 years was also analyzed retrospectively. RSV was found in 21.6% (188/869) of the samples collected from children seen at the emergency department and was found to be more frequent during the autumn, being less frequent or negligible by midwinter. RSV subgroups A and B co-circulated within the same time period in children seen at the emergency department, with varying predominance of either subgroup. There was no significant association of RSV subgroup with disease severity, but only a trend for RSV subgroup B being more frequent in children with risk factors for severe disease.