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Inhibitory effects of antiviral compounds on respiratory syncytial virus replication in vitro.

We examined the inhibitory effect of 20 antiviral compounds, including ribavirin, on the replication of respiratory syncytial virus in HeLa and HEp-2 cell cultures. Of the compounds studied, pyrazofurin and 3-deazaguanine emerged as more potent inhibitors of respiratory syncytial virus than ribavirin. Based on their inhibitory effect on the cytopathogenicity of respiratory syncytial virus in HeLa cells, the average 50% effective dose of pyrazofurin and 3-deazaguanine for eight strains was 0.07 and 1.65 micrograms/ml, respectively; that of ribavirin was 5.82 micrograms/ml. The cytotoxicity of these compounds for HeLa cells was examined by monitoring the incorporation of radiolabeled uridine into cellular RNA. The selectivity indexes of pyrazofurin and 3-deazaguanine exceeded that of ribavirin by 70- and 11-fold, respectively. Pyrazofurin, 3-deazaguanine, and ribavirin inhibited both viral antigen expression and syncytium formation in HeLa cell cultures, as assessed by an indirect immunofluorescence assay. In these assays, pyrazofurin and 3-deazaguanine again proved more potent than ribavirin. 2,5-Diamidinoindole and carbodine were less potent than ribavirin. Various other compounds, i.e., 3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, 3-deazauridine, 3'-C-methyluridine, 5'-deoxy-5-fluorouridine, 5-cyanoimidazole-4-carboxamide, and its ribofuranosyl derivative, did not inhibit the cytopathic effect of the Long strain of respiratory syncytial virus at concentrations greater than or equal to 125 micrograms/ml. Tubercidin, 5-chlorotubercidin, xylotubercidin, neplanocin A, thiosemicarbazone R, and 3-methylquercetine were too toxic to HeLa cells for their inhibitory effects on respiratory syncytial virus to be examined.

Antiviral Agents↗

Indirect haemagglutination test for the detection and assay of antibody to bovine respiratory syncytial virus.

An indirect haemagglutination (IHA) test was used for the rapid assay of antibody to bovine respiratory syncytial virus. Antigens for the sensitisation of formalised tanned erythrocytes were prepared by treatment of virus infected cells with non-ionic detergent. A close serological relationship was shown by the IHA test between the strain of bovine respiratory syncytial virus used and the A2 strain of human respiratory syncytial virus. The IHA test was sensitive and reproducible. A linear correlation was demonstrated between antibody titres obtained by the IHA test and the serum neutralisation test. Titres obtained by the IHA test were approximately 60 times greater than serum neutralisation titres. Serum samples from 803 two-year-old heifers in 48 herds in England were examined by the IHA test. Ninety-four per cent of the animals had antibody to respiratory syncytial virus. Examination of paired serum samples from outbreaks of respiratory disease by the IHA test showed that respiratory syncytial virus was associated with seven out of 15 outbreaks.

Animals↗

Protection against respiratory disease in calves induced by vaccines containing respiratory syncytial virus, parainfluenza type 3 virus, Mycoplasma bovis and M dispar.

A field trial to assess the ability of two vaccines to protect calves against respiratory disease was carried out on a large beef rearing unit in southern England over the two winters of 1983 to 1984 and 1984 to 1985. A quadrivalent vaccine containing the killed antigens of respiratory syncytial virus, parainfluenza virus type 3, Mycoplasma bovis and M dispar or a vaccine containing only the respiratory syncytial virus component were inoculated into 246 and 245 calves, respectively; 245 calves remained as unvaccinated controls. The calves were reared in seven batches and outbreaks of disease occurred in five; significant protection was achieved in the four batches in which disease was associated with respiratory syncytial virus and M bovis infection, together or independently. The death rate from pneumonia was 9 per cent in the control group, 2 per cent in the calves inoculated with the quadrivalent vaccine (P less than 0.001), a protection rate of 77 per cent, and 3 per cent in the calves inoculated with the respiratory syncytial virus vaccine (P less than 0.01), a protection rate of 68 per cent. The proportion of calves receiving treatment for respiratory disease was 38 per cent in the control group, 25 per cent in the calves inoculated with the quadrivalent vaccine (P less than 0.001) and 27 per cent in the calves inoculated with the respiratory syncytial virus vaccine (P less than 0.01). The results show that protection against respiratory disease can be achieved by parenteral vaccination of calves with the appropriate inactivated microorganisms.

Animals↗

Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil.

The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

Adolescent↗

Interferon production in adults with respiratory syncytial viral infection.

Respiratory syncytial virus may cause repeated infections and appreciable illness in adults as well as children. Factors associated with immunity and recovery are poorly understood. We studied 37 adults with natural respiratory syncytial viral illness and eight experimentally infected volunteers for nasal interferon production. Their response was compared to that of 25 adults with influenza. Interferon was detected in only six of those with natural respiratory syncytial virus and in none of those with experimental infection. The quantities of interferon were low (geometric mean, 6 U/mL) and did not appear to affect viral shedding. In contrast, 24 of influenza patients produced interferon and in greater quantities (geometric mean, 116 U/mL). This suggests that interferon is not involved in recovery from respiratory syncytial viral infection and might indicate a lack of a local cell-mediated response that could relate to the often prolonged course and shedding observed with respiratory syncytial virus.

Adult↗

Involvement of antibody, complement and cellular immunity in the pathogenesis of enhanced respiratory syncytial virus disease.

In 1966, infants and children in the USA were immunized with a formalin-inactivated vaccine against respiratory syncytial virus. The vaccine was immunogenic but elicited mainly nonprotective antibody. Upon exposure to respiratory syncytial virus in the community, immunized children developed severe pulmonary disease characterized by bronchoconstriction and pneumonia. Two immunized infants died as toddlers after respiratory syncytial virus infection. Exploration of the mechanisms of disease has dominated the literature for decades. In this review, the pathogenesis of enhanced respiratory disease is discussed and the characteristics of protective and pathogenic respiratory syncytial virus vaccines are examined.

Animals↗

Two novel single amino acid syncytial mutations in the carboxy terminus of glycoprotein B of herpes simplex virus type 1 confer a unique pathogenic phenotype.

We previously reported on a variant of the herpes simplex type 1 (HSV-1) strain 17 syn+, named 17 hep syn, capable of forming giant polykaryocytes (syncytia) in tissue culture and which induced a striking alteration in the pathogenesis of infection in vivo. Following footpad inoculation of mice, 17 hep syn infection resulted in a marked clinicopathologic acute inflammatory response of the inoculated limb and mice died without antecedant limb paralysis typical of the wild-type 17 syn+ infection. The syncytial and pathogenic phenotypes were mapped to a cloned 670-base pair Kpnl-Pstl (0.345-0.351 map units) DNA fragment encoding the carboxy terminal portion of the glycoprotein B (gB). In this report, we focus on the genetics of the region of the 17 hep syn gB gene that conferred both the syncytial and pathogenic phenotypes to 17 syn+. Five 17 syn+ x 17 hep syn syncytial recombinant viruses, R1-R5, generated in marker transfer experiments with cloned 17 hep syn fragments containing gB sequences, produced 17 hep syn-like disease in mice. Sequence analysis of the Kpnl-Pstl fragment of 17 hep syn revealed a single base pair change when compared to the 17 syn+ sequence, predicting an alanine (GCC codon) to valine (GTC codon) amino acid substitution at residue 825 of the mature gB protein, plus loss of an Ncol restriction endonuclease site. Southern blot analysis of Ncol digests of viral DNAs showed that all of the recombinants except R4 contained the same mutation as 17 hep syn. The syncytial phenotype of R4 was, however, mapped to the same region as 17 hep syn and the other recombinants, and the DNA sequence of the 670-base pair Kpnl-Pstl clone of R4 revealed another single base pair change predicting a leucine (CTC codon) to histadine (CAC codon) amino acid substitution at residue 787 of gB. The mutant gBs did not effect viral growth as all of the recombinant viruses had similar in vitro replication kinetics to wild-type HSV-1. These data provide direct evidence that at least two mutations can exist in the carboxy terminus of gB of HSV-1 that promote syncytial formation in vitro and effect pathogenesis in vivo.

Amino Acid Sequence↗

Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2.

Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis.

Amino Acid Sequence↗

Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle.

Skeletal muscle fibers and placental villous trophoblast are the main representatives of syncytia in the human. Both syncytia are derived from fusion of mononucleated stem cells, show a high degree of differentiation, and have lost their generative potency. Consequently, for their growth both depend on fusion of additional stem cells. There is evidence that syncytial fusion is directly or indirectly related to apoptotic events: As early as in the differentiated stages of the mononucleated stem cells, initiation stages of the apoptosis cascade have been observed. After syncytial fusion progression of the cascade is retarded or blocked by a variety of mechanisms. In this review we emphasize the links between apoptosis cascade, differentiation pathways and syncytial fusion. It needs to be elucidated whether these processes simply take place in parallel, both temporally and spatially, or whether there are causal connections between apoptosis cascade and syncytial fusion. Based on recent data obtained for placental villous trophoblast, it is tempting to speculate that early molecular mechanisms of the apoptosis cascade are involved in differentiation and syncytial fusion. Data obtained in skeletal muscles support this assumption and reveal a considerable degree of homology in genesis, maintenance and turnover of both tissues.

Animals↗

The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.

BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions. RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14. CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene.

Amino Acid Sequence↗

The prognostic importance of syncytial growth pattern in medullary carcinoma of the breast.

The histopathological criteria for medullary carcinoma of the breast (MC) used by most pathologists today were delineated by Ridolfi et al. in 1977. The prime criterion is: "A predominantly syncytial growth pattern", predominantly being defined as including 75% or more of the tumour. However, no indication has been given as to why this limit was set at 75%. The present study analyzes the prognostic importance of the extent of syncytial growth in a population of 102 breast cancers with medullary features. Generally, we find a positive prognostic influence of an extensive syncytial growth. The prognostic importance of setting the limit for predominantly syncytial growth at 90% and at 75%, respectively, is evaluated. This comparative study provides no basis for changing the definition of predominantly syncytial growth pattern.

Breast Neoplasms↗

Adrenomedullin is decreased in preeclampsia because of failed response to epidermal growth factor and impaired syncytialization.

To explore the mechanisms of adrenomedullin (ADM) regulation in normal and preeclamptic (PE) states, we determined placental production of ADM and ADM regulation by cytokines. Isolated, purified cytotrophoblast cultures from normal (n=8) and PE (n=10) placentas were cultured for 3 days in the absence or presence of 10 ng/mL epidermal growth factor (EGF), 1 ng/mL transforming growth factor (TGF)-beta1, 10 ng/mL tumor necrosis factor (TNF)-alpha, or 100 U/mL interferon (IFN)-gamma. Cells were also cultured for 3 days in 10% fetal bovine serum for determination of syncytial formation by desmoplakin staining. Pieces of normal and PE placentas were snap-frozen for ADM mRNA measurement. Results showed that basal ADM production into culture medium by radioimmunoassay was significantly lower in PE placental cells. EGF significantly stimulated ADM production in normal trophoblasts but did not in PE placentas. None of the factors TNF-alpha, TGF-beta1, or IFN-gamma altered ADM secretion in either normal or PE placentas. ADM expression by Northern blot analysis demonstrated a 34.3+/-8.3% reduction in mRNA expression in PE placentas. Syncytialization, as assessed by desmoplakin-outlined syncytial units, was decreased in PE placentas (day 3: normal, 16.7+/-1.3%; PE, 5.5+/-2.0%; P<0.01, ANOVA). However, there was a normal increment in syncytialization in response to EGF in normal and PE trophoblast preparations (EGF day 3: normal, 43.8+/-5.6%; PE, 46.1+/-12.3%). We conclude that spontaneous placental syncytialization is impaired in PE and that ADM production is markedly reduced in PE, possibly owing to an impaired EGF response. These abnormalities indicate poor placental production of ADM as the likely cause of a failed compensatory increase in maternal serum ADM levels in PE.

Adrenomedullin↗

A syncytium regression test to detect antibodies to bovine syncytial virus.

A test to detect antibodies to bovine syncytial virus was developed from the observation that syncytia in monolayer cell cultures infected with bovine syncytial virus regress and disappear in the presence of bovine syncytial virus antibodies. The test is useful in monitoring the presence of bovine syncytial virus and bovine syncytial virus antibodies in cattle used in studies on bovine leukosis.

Animals↗

Economic analyses of respiratory syncytial virus immunoprophylaxis in high-risk infants: a systematic review.

OBJECTIVE: To systematically review all published economic analyses of the only 2 available agents for respiratory syncytial virus immunoprophylaxis in high-risk infants: respiratory syncytial virus immunoglobulin intravenous and palivizumab. DATA SOURCES: Economic evaluations of respiratory syncytial virus immunoprophylactic agents were identified from the MEDLINE and HealthSTAR databases using various combinations of the following search terms: respiratory syncytial virus immunoglobulin intravenous, palivizumab, cost, and cost-effectiveness. The search was limited to articles published in English between January 1, 1990, and August 31, 2001. Additional studies were obtained by searching bibliographies of all relevant identified articles. STUDY SELECTION: Only studies that performed an economic analysis of either or both of these agents in an infant population were included. Letters to the editor and commentaries that included informal economic analyses were excluded. Twelve of the 21 identified studies met the selection criteria. DATA EXTRACTION: Two of us (S.K.-B. and J.D.) independently reviewed the articles and extracted summary information using a standardized abstraction form, with differences resolved by consensus. DATA SYNTHESIS: Estimates ranging from cost savings to considerable incremental costs per hospitalization avoided with use of either agent were observed across studies. Studies comparing the 2 agents reported mixed results about their relative cost-effectiveness in different infant subgroups. The divergent results may be explained by differences in the study methods and assumptions, but they also reflect the poor quality of some of the economic analyses. CONCLUSION: In light of the issues identified in this review, providers, payers, and health policymakers need to critically appraise and judiciously interpret cost-effectiveness research on these agents.

Antibodies, Monoclonal↗

The European Forum for Clinical Management: prophylaxis against the respiratory syncytial virus in infants and young children with congenital cardiac disease.

A recent, randomised, double-blind, placebo-controlled trial has demonstrated the effectiveness of palivizumab (Synagis) for prophylaxis against infection by the respiratory syncytial virus in 1,287 young children with congenital cardiac disease. Guidelines for the use of palivizumab in these children considered to be at high risk were published by the American Academy of Pediatrics, followed by similar guidelines from the British Paediatric Cardiac Association, and recommendations from a number of other individual countries, including Canada, Germany, Spain, and France. In May, 2004, further discussion was held between a group of 15 paediatric cardiologists at a European forum for clinical management held in Munich, Germany. The objective of this forum was to define optimal recommendations on prevention of infection by the respiratory syncytial virus in infants and young children with congenital cardiac disease, appropriate to the clinical needs and style of those practising in paediatrics in individual countries. Participants were invited because of their knowledge of the therapeutic area, and for their experience of using palivizumab for prophylaxis against the respiratory syncytial virus in children with congenitally malformed hearts. Measures to educate the carers of children with such congenital malformations on precautions against infection by the respiratory syncytial virus were discussed, along with the many different aspects of best practice for therapeutic prophylaxis with palivizumab. The most appropriate timing of prophylaxis, recommendations for which children are most likely to benefit from prophylaxis, and suggested protocols were among the issues covered. The recommendations resulting from the discussions are presented in this paper, as a step towards reaching consensus.

Antibodies, Monoclonal↗

Nasal and otologic effects of experimental respiratory syncytial virus infection in adults.

PURPOSE: Upper respiratory tract viral infections continue to cause substantial patient morbidity and complications including sinusitis, otitis media, and pneumonia. This study was conducted to more clearly define the extent and frequency of nasal and otologic effects of respiratory syncytial virus infection in healthy adult volunteers. MATERIALS AND METHODS: Thirty-two healthy, susceptible adult volunteers were cloistered for a 9-day period. During this time, subjects underwent nasal inoculation with respiratory syncytial virus. Monitoring included daily self-assessment of general health, as well as nasal and otologic symptoms. Objective measurements of nasal and otologic function included expelled nasal secretion weight, saccharin-dye mucociliary clearance, sonotubometry, tympanometry, and physical examination. A nasal lavage was done each morning, and fluids were submitted for viral detection. Twenty-one days after viral inoculation, all subjects had convalescent blood samples drawn for assay of virus-specific antibody titres. RESULTS: Eighteen subjects (56%) became infected with the challenge as determined by either viral shedding (47%) or antigen detection (41%) from the nose or a 4-fold rise in virus-specific antibody titer (34%). Infected subjects more frequently reported adverse nasal (congestion, rhinorrhea) and general symptoms (fever, malaise, illness). By day 6, only 46% of infected subjects had normal middle ear pressure (> -100 mm H2O). Nasal secretion also increased substantially after infection. No patterned changes in mucociliary clearance or sonotubometry occurred, and no subjects developed otitis media. CONCLUSIONS: Nasal inoculation of healthy, susceptible adult volunteers with respiratory syncytial virus results in detectable infection in only about half of the subjects challenged. Infected subjects experience substantial symptoms and signs of a viral upper respiratory tract illness. As in our previous studies using rhinovirus and influenza A virus, respiratory syncytial virus disrupted the maintenance of normal middle ear pressures. These findings provide further support for the relationship between viral upper respiratory tract infections and otitis media.

Adolescent↗

Persistence of respiratory syncytial virus (RSV) infection and development of RSV-specific IgG1 response in a guinea-pig model of acute bronchiolitis.

Acute respiratory syncytial virus (RSV) bronchiolitis in children can result in sequelae of recurrent wheezing and asthma and production of RSV-specific immunoglobulin E (IgE), but the pathogenesis of these sequeleae is poorly understood. Guinea-pigs experimentally inoculated with human RSV show histological evidence of acute bronchiolitis and chronic persistence of viral antigens and genome in the lungs; whether this persistence is due to infectious replicating virus, and whether infected animals develop RSV-specific immunoglobulin G1 (IgG1) (the main class of antibody involved in guinea-pig allergic responses) is unknown. Guinea-pigs were inoculated intranasally with human RSV or with uninfected cell culture supernatant. At times ranging 1-60 days postinoculation, the viral titre in the lung was determined by immunoplaque assay (a method combining viral culture and immunocytochemistry). Serum titres of RSV-specific IgG1 antibodies were determined by enzyme-linked immunosorbent assay. Bronchiolar inflammation was assessed on coded lung sections, by using a semiquantitative, histological scoring system based on features of human acute bronchiolitis. Infectious RSV was cultured from the lungs of infected animals on all study days, with maximal viral replication observed on Day 3. RSV-specific IgG1 antibodies were detected in all RSV-inoculated animals from Day 7 onward, with the highest antibody titre measured on Day 28. RSV-inoculated guinea-pigs had maximal bronchiolar inflammation on Day 7, and had significantly increased polymorphonuclear cell infiltrates on Days 28 and 60. Respiratory syncytial virus chronically persists as infectious virus in the guinea-pig lung. Infected animals develop an anti-respiratory syncytial virus immunoglobulin G1 antibody response, histological evidence of acute bronchiolitis, and chronic airway inflammation. Persistent respiratory syncytial virus lung infection may be important in the pathogenesis of postbronchiolitis wheezing and asthma in children.

Acute Disease↗

Palivizumab in the prophylaxis of respiratory syncytial virus infection.

Respiratory syncytial virus infection continues to be one of the most important health problems in infancy. Active prophylaxis against this infection (i.e., vaccination) is not available. Therefore, protection of high-risk infants is possible only by passive prophylaxis with specific antibodies. Palivizumab (Synagis) and respiratory syncytial virus intravenous immune globulin are licensed by the US Food and Drug Administration for the prevention of severe lower respiratory tract infections caused by respiratory syncytial virus in infants with bronchopulmonary dysplasia, infants with a history of premature birth (< or =35 weeks gestational age) and children with hemodynamically significant congenital heart disease. Palivizumab is a humanized monoclonal antibody produced by recombinant DNA technology, directed to an epitope in the A antigenic side of the F-protein of the respiratory syncytial virus. This review discusses the characteristics of this drug in detail.

Animals↗