Animal models of respiratory syncytial virus infection

Over the past two decades, animal models of respiratory syncytial virus (RSV) infection have been developed using primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters. Use of these models has shed light on the mechanisms of vaccine-enhanced disease seen in clinical trials of a formalin-inactivated RSV vaccine and has provided a means for testing efficacy and safety of candidate prophylactic and therapeutic strategies. The development of multiple animal models has coincided with the realization that RSV disease in humans is a multifaceted disease whose clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status, and concurrent disease within subpopulations. There is no single human subpopulation in whom all forms of RSV disease manifest, nor is there a single animal model that duplicates all forms of RSV disease. The choice of an experimental model will be governed by the specific manifestation of disease to be studied.     

Immunobiology of bovine respiratory syncytial virus infections

This paper describes recent findings on the immunobiology of bovine respiratory syncytial virus (BRSV) infections. The pathobiology of alveolar macrophages and BRSV, and the immunological reaction of cattle to the virus after natural or experimental infection, or vaccination, were studied. Because in severe cases BRSV infection leads to lower respiratory tract disease, replication of BRSV in alveolar macrophages was studied. Alveolar macrophages, which are important aspecific defense cells in the lower respiratory tract, exhibited a high intrinsic resistance to BRSV. Furthermore, BRSV-infected alveolar macrophages produced significantly less nitric oxide (which has a bacteriocidal effect) than uninfected macrophages. The kinetics of antibody titres against the envelope protein G were different from those of antibody titres against the envelope protein F. For example, many animals that are reinfected do not possess antibodies against the G protein. After vaccination or after natural infection, antibody titres against the F and G protein, and against epitopes on the F protein, differed markedly, and also in animals with different MHC haplotypes. These findings may be related to differences in protection. The strains of BRSV that circulate in the Netherlands belong to the subgroups A and AB. There was no evidence for differences in virulence between these subgroups. BRSV could be detected in 30% of lungs obtained from calves suffering from severe lower respiratory tract disease. Based on cross-protection studies, calves that were infected with a virus from a particular BRSV subgroup were protected against reinfection with a virus from a different subgroup. A recombinant gE-protein negative bovine herpesvirus 1 vaccine carrying a gene encoding the G protein of BRSV, and a DNA vaccine encoding the same protein afforded protection after experimental challenge of calves. This offers the possibility to develop effective multivalent (gE-negative BHV1) marker vaccines in the future.     

Distinct patterns of T- and B-cell immunity to respiratory syncytial virus induced by individual viral proteins

The purpose of this study was to determine the safety of 4 week re-use of vinyl urinary leg and bed bags in the acute rehabilitation setting when decontaminated daily with a dilute bleach (sodium hypochlorite) rinse. Patients requiring urinary bags (n = 54) were randomly assigned to Control (C) and Experimental (E) groups. C's bags were replaced weekly; E's only after four weeks. Both groups received identical daily bag decontamination and weekly urine and bag cultures. No significant differences were found between groups with ANCOVA, controlling for baseline urine cultures, age, number of days catheterized, and use of antibiotics. Thirty different organisms were cultured in urine and bags; when the procedure was done daily, all bag cultures showed only minimal contamination (0-100CFU/mL). Bacterial growth (4.4% of leg bags) > 100CFU/mL was found only when the daily decontamination procedure had been omitted. In fact, 57% of leg bags and 76.5% of bed bags returned with no growth. We conclude that it is safe and cost effective to reuse vinyl urinary leg and bed bags for four weeks.     

Inactivation of respiratory syncytial virus by detergents and disinfectants

The activity of a number of detergents and disinfectants against respiratory syncytial virus (RSV) was evaluated in an in vitro assay system. Equal volumes of RSV and serial 10-fold dilutions of the test agents were mixed at 4 degrees C for 5 minutes. The RSV titer in each mixture was compared with that of untreated RSV alone. In 14 experiments with input RSV titers ranging from 2.6 x 10(3) to 2 x 10(7) plaque-forming units/ml, a 10-fold dilution of 5.25% sodium hypochlorite (generic bleach) inactivated (> or = 3-log reduction in titer) the virus. With lower RSV titers inactivation was also observed at a 100-fold dilution of bleach. Fetal calf serum concentrations up to 50% as an organic load did not diminish the bleach effect. The degree of RSV inactivation was also defined for Lysol, povidone-iodine, Amphyl, Hibiclens, Osyl, ethanol and Listermint. The short contact time, the reproducible nature of the findings and the continued effectiveness with increasing organic loads all suggest that detergents and disinfectants can potentially play an important role in decreasing the spread of RSV infection.     

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a fourfold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.     

Phosphoprotein profile analysis of ruminant respiratory syncytial virus isolates

OBJECTIVE: To determine the apparent molecular weight for 24 ruminant respiratory syncytial viruses (RSV) on the basis of differences in the electrophoretic mobility of the phosphoprotein (P protein). PROCEDURE: 29 bovine RSV (BRSV), 20 of which were not previously tested, 3 ovine RSV, and 1 caprine RSV isolates were selected for determination of electrophoretic mobility of the P protein. Virus radiolabeled with [35S]methionine was immunoprecipitated with polyclonal antiserum to BRSV and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: On the basis of apparent molecular size of the P protein, all isolates could be categorized into 2 electropherotypes, low molecular size of 36 kd and high molecular size of 38 kd. Twenty-three BRSV, the 3 ovine RSV, and 1 caprine RSV isolates had a high molecular size P protein; 6 BRSV isolates had a low molecular size P protein. CONCLUSIONS: The apparent molecular size of the P protein of the ruminant RSV strains is greater than that of the human RSV subgroups, providing further evidence of their distinctiveness. Whether categorization of electrophoretic mobility of the P protein of BRSV underlies distinct antigenic subgroups, as it does in human RSV, requires further antigenic and genetic analysis. CLINICAL RELEVANCE: Antigenic subgroups of ruminant RSV may have relevance in the development of new vaccines for control of the disease.     

Antigenic structure of human respiratory syncytial virus fusion glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

Antigenic and genomic diversity of central European respiratory syncytial virus strains

Thirty-two RSV strains recovered during the winter months of 1987/88 to 1993/94 from hospitalized children in Vienna, Austria and Zagreb, Croatia were analysed for antigenic and genetic variations. Twenty-nine of the 32 isolates investigated belonged to group A and 3 to group B, with the majority of infections caused by subgroup A1 (21 of 29). Restriction endonuclease mapping of PCR products derived from parts of the N and G gene of 18 group A strains identified 3 distinct lineages, very similar to those defined by analysis of recurrent epidemics in Birmingham, United Kingdom during the same period. Results of this study provide further information on the global pattern of RSV and show that very similar viruses are present simultaneously in widely separated areas.     

Effect of respiratory syncytial virus infection of HeLa-cell macromolecular synthesis

Cells infected with respiratory syncytial (RS) virus eventually die but there appears to be no specific mechanism for shutting off cellular synthesis of macromolecules. DNA and RNA synthesis, as measured by the incorporation of labelled thymidine or uridine, do not begin to shut down until some time between 11 and 18 h after infection. By 18 h their rates of synthesis are reduced to approx. 50% for DNA and 35% for RNA. Protein synthesis continues throughout the course of infection at approximately the same rate. Synthesis of most of the cellular polypeptides also continues, but the distribution of polypeptides of high and low mol. wt. shifts. The increase in the proportion of those of high mol. wt. includes a peak that represents one of the seven previously identified virion polypeptides. Another consequence of RS virus infection is an increase in glucosamine incorporation, beginning near the end of the virus eclipse period (12 h after infection), which may be associated with virion glycoprotein synthesis. Polyacrylamide-gel electrophoresis of glucosamine-labelled cells reveals that at 18 h after infection two of the three previously identified virion glycoproteins are present.     

Detection of antibody to respiratory syncytial virus by membrane fluorescence

An indirect membrane fluorescent antibody technique was established to study HEp 2 cells infected with respiratory syncytial (RS) virus. It was possible to detect IgG and IgM antibody to RS virus in the sera of patients with respiratory infections using this technique. The technique was further applied to the detection of IgA antibody to the same virus in colostrum.     

A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

A critical issue has been the observation that vaccination of children with a formalin-inactivated respiratory syncytial virus (RSV) vaccine is associated with disease enhancement. We have taken advantage of bovine RSV and our experience with this disease in calves to develop a natural model that parallels human disease. Using formalin-inactivated bovine RSV vaccine calves were either sham-vaccinated/infected, vaccinated/infected, or vaccinated/sham-infected and their clinical signs, pulmonary function, and histological lung lesions quantitatively scored. Interestingly there was significantly greater disease in vaccinated/infected calves and histological lesions in calves were similar to those of affected children. Finally, we note that vaccination did not induce neutralizing antibodies, but IgG antibodies were detected by ELISA. Our model of RSV enhanced disease is important because it provides quantifiable evidence of disease severity that can be applied to evaluate the mechanisms of immunopathology and the safety of candidate RSV vaccines.     

Nucleotide sequence analysis of the ovine respiratory syncytial virus G glycoprotein gene

Respiratory syncytial virus (RSV) infects humans and animals including ruminants. Among the 10 genes coded for in the viral genome, the putative attachment glycoprotein G gene has been the most variable among strains. Human RSV have been divided to two subgroups based on immunological and base sequence data on the attachment glycoprotein G and its gene, respectively. It has been suggested that similar antigenic diversity also exists among bovine RSV (BRSV) isolates. In this study, we report on the cloning and sequencing of the G glycoprotein from an ovine RSV (ORSV) strain originally isolated from a naturally infected sheep with rhinitis. This ORSV G glycoprotein gene had greater identity to the BRSV G gene than to the human RSV G gene. ORSV G gene and its encoded protein shared 70 and 62% nucleotide and amino acid identity to the equivalent gene and encoded protein, respectively, of BRSV but, in contrast, only 50-55% and 21-29% identity, respectively, to equivalent sequences of the HRSV strains. The relationship of the ORSV to other RSV subgroups and the possibility that ORSV could be a subgroup of the ruminant RSV is discussed.     

Antigenic and nucleic acid analysis of nosocomial isolates of respiratory syncytial virus

Monoclonal antibodies and ribonuclease protection were used to analyze antigenic and genomic diversity among 42 isolates of group A respiratory syncytial virus (RSV) from studies of nosocomial RSV carried out at the University of Rochester during the 1974-1975 and 1975-1976 RSV seasons. Three distinct subgroups or lineages and a total of 12 viral variants were present. Against this background of diversity, an outbreak was recognized that included 13 indistinguishable isolates occurring during a 2-week period. This outbreak accounted for 6 of the 8 infants with nosocomial infection. In contrast to the limited diversity of the nosocomial isolates, isolates from the 10 infants with community-acquired infection included 8 variants. Like those from community outbreaks of RSV, isolates of RSV from hospitalized patients are virologically heterogeneous. However, discrete outbreaks associated with transmission of a single strain can occur.     

Sequential respiratory syncytial virus and cytomegalovirus pneumonia following bone marrow transplantation

A 6-month-old child with familial hemophagocytic lymphohistiocytosis (FHL) experienced early sequential pneumonia due to respiratory syncytial virus (RSV) and cytomegalovirus (CMV) following bone marrow transplantation (BMT). The patient was deficient in natural killer (NK) cell activity (as found frequently in patients with FHL), and this risk factor may have played a major role in the concomitant infection by the two viral pathogens. Rapid diagnostic methods for both viruses are essential and early specific treatment may serve to ameliorate RSV- and CMV-induced lung injury in these life-threatening infections.     

Further characterization of the complementation group B temperature-sensitive mutant of respiratory syncytial virus

ts-2, a temperature-sensitive and plaque morphology mutant of respiratory syncytial virus and sole representative of complementation group B, was compared with members of the other complementation groups of respiratory syncytial virus (group A [ts-1] and group C [ts-7]). ts-2 was found to be 10- to 1,000-fold more restricted in growth and ability to spread at restrictive temperatures (37, 38, and 39 degrees C) than at the permissive temperature (32 degrees C). In temperature shift-up experiments, the ts defect of ts-1 and other members of complementation group A was found to effect a late function that was required for at least 13 h in the replicative cycle. The ts lesion of ts-7 affected a function early in the replication cycle. In contrast, ts-2 was not temperature sensitive when studied by the shift-up technique. The discrepancy between the ts plaque property and failure to detect temperature sensitivity during the shift-up experiment was resolved when it was shown that ts-2 had a defect in adsorption or penetration or both at the restrictive temperature. Clonal analysis of revertant ts-2 showed a coordinate restoration of ts+ phenotype ans syncytium-forming capacity. It appears that ts-2 has a defect in a protein that is involved in adsorption and/or penetration of virus and is also responsible for cell fusion activity.     

Preparation of respiratory syncytial virus subgroup A and B antigens for enzyme immunoassay antibody detection

A simplified method was described for purification of respiratory syncytial virus (RSV) subgroup A and B aimed to be used as antigens in enzyme immunoassay (EIA). The titer of each RSV subgroup and the amount of protein was determined from the visible band in 45% sucrose gradient. The quality of prepared RSV subgroup antigens for EIA was described in terms of the achievable final titer, the amount of protein, and EIA criss-cross titration. The RSV subgroup A and B antigens, diluted as 1:100 (low opalescent band in 45% sucrose layer) or 1:800 (high opalescent band in 45% sucrose layer) produced a positive reaction in EIA criss-cross titration with IgG antibodies from the patient's serum (convalescent phase) diluted as 1:25,600 (for RSV A) and 1:6,400 (for RSV B). This method offers shorter and more simplified steps of viral antigen purification, and provides acceptable quantity and quality of viral antigens appropriate for use in EIA.     

Epidemiology of respiratory syncytial virus infection requiring hospitalization in East Denmark

BACKGROUND: Prophylaxis against infection caused by respiratory syncytial virus (RSV) with high titered RSV immunoglobulin or humanized antibody may soon be available in Europe. OBJECTIVE: To study the epidemiology of RSV infections requiring hospitalization in infants <6 months in East Denmark to provide a rational basis for decisions concerning prophylaxis against RSV. METHOD: Populat ion-based retrospective review of case records of infants <6 months admitted to pediatric departments with RSV infection in East Denmark from November 1, 1995, to April 30, 1996. RESULTS: Data were obtained from 459 infants. Seventy-three had predisposing conditions: prematurity, 49; pulmonary disease, 2; congenital heart disease, 7; neurologic disease, 6; others, 9. One preterm infant had bronchopulmonary dysplasia. The incidence of RSV infection requiring hospitalization in East Denmark among infants <6 months was estimated to be 34/1000/season. It was 32/1000/season among term infants and 66/ 1000/season among preterm infants (P<0.001). Infants with predisposing conditions and/or nosocomial infection (n = 24) had significantly more severe courses than otherwise healthy infants (P<0.01). One-hundred thirty infants received respiratory support by nasal continuous positive airway pressure, but only six required mechanical ventilation. No infants died. CONCLUSION: The course of RSV disease in East Denmark was milder than reported elsewhere, possibly as a result of the low prevalence of bronchopulmonary dysplasia in Denmark. However, RSV constitutes a considerable burden to the Danish pediatric health care system, and therefore prophylaxis against RSV is desirable.