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PubMed2001

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides.

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.

Animals↗
PubMed2001

Epithelial cells infected with respiratory syncytial virus are resistant to the anti-inflammatory effects of hydrocortisone.

In this work we continue our study of the biochemical responses of respiratory epithelial cells to infection with human paramyxovirus pathogens. In our earlier studies, we detected elevated concentrations of the proinflammatory chemokines MIP-1alpha and IL-8 in upper and lower respiratory tract secretions from patients infected with respiratory syncytial virus (RSV). Here we demonstrate the same trend for individuals infected with parainfluenza virus (PIV), with elevated concentrations of MIP-1alpha and IL-8 (means of 309 +/- 51 and 2280 +/- 440 pg/ml/mg protein, respectively) detected in nasal wash samples from 17 patients with culture-positive PIV. Similar to our findings with RSV, cells of the HEp-2 epithelial line and primary cultures of human bronchial epithelial cells respond to PIV infection with production and release of both MIP-1alpha and IL-8. Addition of the glucocorticoid anti-inflammatory agent hydrocortisone (200-1000 ng/ml) attenuated the production of MIP-1alpha and IL-8 in PIV-infected cells while having minimal to no effect on the production of these mediators from cells infected with RSV. Neither virus infection resulted in a change in the total cellular concentration of glucocorticoid receptors, nor did hydrocortisone exert any differential effect on viral replication. As repression of chemokine production by epithelial cells is likely to result in diminished recruitment of proinflammatory leukocytes, these results may explain in part why glucocorticoid therapy reduces the symptoms associated with acute PIV infection, but have little to no effect in the overall outcome in the case of RSV.

Anti-Inflammatory Agents↗
PubMed2002

Chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis.

This study measured chemokines in nasal lavage fluids (NLF) from infants with respiratory syncytial virus (RSV) bronchiolitis, defined by lung hyperinflation and wheezing. Comparison was made to RSV-positive infants without bronchiolitis and RSV-negative infants with acute respiratory illnesses. RSV-positive illnesses were associated with increased epithelial shedding, increased RANTES/protein ratios, and increased IL-8/protein ratios in NLF compared to RSV-negative illnesses. Among RSV-positive infants, bronchiolitics had greater total cell counts and percentage epithelial cells in NLF than nonbronchiolitics. Bronchiolitics also had roughly twice the NLF RANTES/IL-8 ratio than nonbronchiolitics (P =.043). Semiquantitative reverse transcriptase-polymerase chain reaction of nasal epithelium suggested similar RANTES/IL-8 ratio increases among bronchiolitics. A more mildly affected, RSV-positive outpatient population showed none of these differences. We conclude that RSV bronchiolitis is associated with a shift toward relatively more RANTES in nasal secretions of infants sick enough to require hospitalization, and mucosal epithelium may contribute to this process. Similar processes in the lower airways may enhance inflammation due to RANTES-responsive cell types and affect clinical manifestations.

Blood Proteins↗
PubMed1994

Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Amino Acid Sequence↗
PubMed1997

Induction of protective immunity in rodents by vaccination with a prokaryotically expressed recombinant fusion protein containing a respiratory syncytial virus G protein fragment.

A subunit approach to the development of a respiratory syncytial virus (RSV) vaccine was investigated. It involved the production, in Escherichia coli, of an RSV (Long) G protein fragment (G2Na) as a C-terminal fusion partner to an albumin binding region (BB) of streptococcal protein G. G2Na incorporated amino acid residues 130-230 and was specifically recognized by murine anti-RSV-A polyclonal serum. In mice, intraperitoneal immunization with BBG2Na induced high anti-RSV-A serum ELISA titers and low to moderate neutralization activity. The immune response induced by BBG2Na demonstrated a potent protective efficacy against upper and lower respiratory tract RSV-A infection. The immunogenicity and protective efficacy of BBG2Na was maintained for at least 47 and 48 weeks, respectively, and was as potent and durable as live RSV-A administered in a similar fashion. Intramuscular immunization of cotton rats with BBG2Na protected lungs from both homologous and heterologous virus challenge. In contrast to mice, however, cotton rat nasal tracts were not protected after BBG2Na immunization. Consistent with antibody-mediated protection, virus was cleared within 24 hr from the lungs of BBG2Na-immunized mice. The anti-RSV-A antibodies induced in mice were exclusively of the IgG1 isotype and were detected in the serum, lungs, and nasal tracts. Passive transfer of these antibodies prevented acute, and eliminated chronic, RSV-A lung infection in normal and immunodeficient mice, respectively, confirming that such antibodies are important and sufficient for BBG2Na-induced pulmonary protection. Our results clearly demonstrate that BBG2Na contains an important immunogenic domain of the RSV G protein. The prokaryotic origin of this protein indicates that glycosylation of the RSV G protein is not necessary for protective efficacy. Thus, BBG2Na has potential as an RSV subunit vaccine.

Animals↗
PubMed1997

Protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus.

We investigated whether a recombinant bacteriophage displaying a disease-specific protective epitope could be experimentally used as a vaccine to confer protection of immunized animals against infection. We genetically engineered a recombinant phage, fd, displaying at its surface a chimeric pIII coat protein fused to the previously identified protective epitope 173-187 from the glycoprotein G of the human respiratory syncytial virus (RSV). A selected recombinant fd phage elicited a strong immune response in mice, inducing a high level of circulating RSV-specific antibodies. Mice immunized with the recombinant phage acquired a complete resistance to RSV infection as evidenced by the lack of detectable virus particles in their lungs following intranasal challenge with live RSV. In contrast, a high level of virus particles was found in the lungs of either animals immunized with the wild-type fd phage or nonimmunized mice. To our knowledge, this is the first study to report the ability of a phage presenting an immunogenic peptide to prevent infection of immunized animals by a pathogen. This finding should facilitate the identification of pathogen-specific protective epitopes selected from random phage peptide libraries, as it is simpler and less expensive than the conventional method of synthesis and coupling of phage-specific peptide ligand sequences for immunization.

Amino Acid Sequence↗
PubMed1999

Induction of CD95 (Fas) and apoptosis in respiratory epithelial cell cultures following respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation. In mouse models, and in immunosuppressed patients, CD8(+) T cells are necessary for RSV clearance. In vitro, RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8(+) T cells in initiating apoptosis of the target cell. One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8(+) T lymphocytes and Fas expressed on the target cell. In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549) in vitro. Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs. RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas. These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this process in vivo.

Animals↗
PubMed2000

Plasmid DNA encoding the respiratory syncytial virus G protein is a promising vaccine candidate.

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory diseases in infants, young children, and the elderly. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV preparation. Enhanced lung disease was induced by the vaccination post-RSV exposure. Previous studies in mice primed with RSV G protein either formulated in adjuvants or delivered by recombinant vaccinia viruses have indicated that enhanced lung pathology resulted from a Th2-type host immune response against the viral G protein. However, in the present report, we have demonstrated that vaccination with plasmid vectors encoding either a full-length or a secreted G protein (DNA-G) clearly elicited balanced systemic and pulmonary Th1/Th2 cytokine responses in mice and did not induce an atypical pulmonary inflammatory reaction post-RSV challenge in cotton rats. DNA-G immunization also induced marked virus neutralizing antibody responses and protection against RSV infection of the lower respiratory tract of both mice and cotton rats. So far, only genetic immunization has been able to induce a balanced Th1/Th2 response with the RSV G protein, reminiscent of that induced by live RSV. Therefore, DNA-G is a promising immunogen for inclusion in a nucleic acid RSV vaccine.

Animals↗
PubMed2000

Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo.

Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: DeltaNS1NS2, DeltaM2-2SH, DeltaM2-2NS2, DeltaSHNS1, DeltaSHNS2, and DeltaSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, DeltaM2-2NS2 was most attenuated. DeltaM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log(10) compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication.

Animals↗
PubMed2001

RhoA is activated during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants and immunocompromised adults. We have recently shown the RSV F glycoprotein, which mediates viral fusion and entry, interacts with the cellular protein RhoA in two-hybrid and in vitro binding assays. Whether this interaction occurs in living cells remains an open question. However, because RhoA signaling is associated with many cellular functions relevant to RSV pathogenesis such as actin cytoskeleton organization, expression of proinflammatory cytokines, and smooth muscle contraction, we asked whether RhoA activation occurred during RSV infection of HEp-2 cells. We found that the amount of isoprenylated and membrane-bound RhoA in RSV-infected cultures was increased. Further evidence of RhoA activation was demonstrated by downstream signaling activity mediated by RhoA. There was an increase in p130(cas) phosphorylation during RSV infection, which was prevented by Y-27632, a specific inhibitor of Rho kinase, or lovastatin, an HMG-CoA reductase inhibitor that reduces the synthesis of groups needed for isoprenylation. In addition, RSV infection of HEp-2 cells resulted in an increase in the formation of actin stress fibers. Pretreatment of HEp-2 cells with Clostridium botulinum C3 exotoxin, an enzyme that specifically ADP-ribosylates and inactivates RhoA, prevented RSV-induced stress fiber formation. These observations indicate that RhoA and subsequent downstream signaling events are activated during RSV infection, which has implications for RSV pathogenesis.

Actins↗
PubMed2001

Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.

The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.

Amino Acid Sequence↗
PubMed2002

Targeted therapy of respiratory syncytial virus in African green monkeys by intranasally administered 2-5A antisense.

Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2prime prime or minute-5prime prime or minute-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2prime prime or minute-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log(10) units in a dose- and time-dependent manner.

Adenine Nucleotides↗
PubMed2002

Passive transfer of serum antibodies induced by BBG2Na, a subunit vaccine, in the elderly protects SCID mouse lungs against respiratory syncytial virus challenge.

Respiratory syncytial virus (RSV) is responsible for severe low respiratory tract infections in young infants and the elderly. To investigate whether BBG2Na, a recombinant subunit vaccine comprising aa 130-230 of the RSV G protein, induced protective Abs in subjects over 60 years during phase II clinical trial, pre- and postimmunization sera of individuals immunized with BBG2Na or placebo were transferred into SCID mice before RSV challenge. These sera dose-dependently reduced lung RSV titers. However at some points of serial dilutions, postimmunization sera of BBG2Na-immunized subjects only were significantly more efficient than the corresponding preimmunization sera, in agreement with the induction of an increased Ab response against multiple epitopes on RSV-A G protein. Thus, BBG2Na is immunogenic in the elderly and confers passive protection in mice after serum transfer. To our knowledge, this is the first description of protective Abs induced by a subunit vaccine in human.

Administration, Intranasal↗
PubMed1996

Respiratory syncytial virus infections in pediatric renal transplant recipients.

Immunocompromised patients are considered at increased risk from respiratory syncytial virus (RSV) infection. We examined the incidence and outcome of RSV infection in pediatric renal transplant (Tx) recipients on chronic immunosuppressive therapy. Of 173 recipients transplanted between November 1985 and April 1993, 5 (3%) developed RSV infection (age range 11-39 months). Initial immunosuppression included prednisone, azathioprine, cyclosporine, and polyclonal antibody therapy. Time from Tx to onset of RSV infection was 1 day to 7 months. Symptoms included rhinorrhea, cough, tachypnea, retractions, fever, wheezing, and abnormal chest X-ray. Treatment included bronchodilator therapy, bronchial drainage, ribavirin, and mist tent. Azathioprine was transiently withheld for leukopenia during treatment in 2 recipients. Three recipients developed biopsy-proven acute rejection during (n = 2) or immediately following (n = 1) RSV infection; all responded to corticosteroid treatment. RSV infection is not commonly diagnosed in pediatric renal Tx recipients. The course of RSV infection in our patients did not differ from that reported in normal children. The possible association between RSV and acute rejection warrants further observation. When diagnosed early, RSV infection does not appear to be associated with increased mortality in pediatric renal Tx recipients. Larger numbers of recipients need to be studied to confirm these results.

Child, Preschool↗
PubMed1993

Dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds.

To study the epidemiology of respiratory syncytial virus (RSV) infections during the year, the incidences of primary infections and reinfections were monitored by titrating antibodies to bovine RSV (BRSV) in cattle above 2 months of age in 6 dairy herds in the Netherlands. From August 1990 until September 1991, 884 cattle were sampled at one-month intervals. A total of 155 cattle, most under two years of age, had a primary antibody response. Antibody rises were found in 259 cattle of all ages. The highest incidences of BRSV infections were found in one period either in autumn or winter. In other seasons, primary infections were rare, whereas reinfections were not uncommon. In 5 out of the 6 herds, two seronegative sentinel calves were introduced at the end of the winter and none developed specific antibodies before the next winter. The observations strongly suggest that, in spite of regular reinfections, BRSV circulates during spring or summer at a very low level or not at all. Persistent BRSV infection in a number of cows might be a means for the virus to survive during summer, but a steady rate of reinfection of seropositive cows throughout the year at a low level might also maintain a reservoir of infectious virus. This study adds to the knowledge of frequency and timings of primary infections and reinfections of BRSV and it might contribute to the study of these issues of human RSV.

Animals↗
PubMed1994

Subgrouping of respiratory syncytial virus strains from Australia and Papua New Guinea by biological and antigenic characteristics.

Strains of respiratory syncytial virus from 3 major areas of Australia and Papua New Guinea (PNG) were analyzed for variations in their antigenic and biological properties and in the molecular weights of their major structural proteins. Seventy-eight strains from infants and young children with LRI were collected from 1981-1984. The RSV season in the Australian cities lasted from April through September, with major peaks in July of each year, while the RSV season in tropical PNG was year-round, with small peaks in March and October of each year coinciding with excessive rainfall. Fifty-six strains were analyzed in detail; 40 were typed by time-resolved fluoroimmunoassay with monoclonal antibodies as group A strains and 16 were group B; both groups were concurrent. Three children of one family had sequential RSV infections 13 months apart, and the etiologic group A strain was identical both years in terms of growth and antigenic properties with strain-specific ferret antisera; the second infection was milder in all three children. On average, the group A strains replicated considerably better than group B strains in HEp2 cells, producing 53% more syncytia and 99% higher infectious virus titers in 31% less time in culture. Ten group A and B reference strains exhibited the same growth patterns as the A and B regional strains, respectively. Differences in antigenicity as measured with hyperimmune antisera to prototype Long strain were even greater. Group A strains exhibited a mean 68% greater IFA staining than B strains, a 71% greater EIA reaction, and were neutralized to 69% higher serum titers than B strains. Again, the reference A and B strains included as controls gave patterns identical to those of the regional strains. Finally, the P phosphoprotein had consistently higher molecular weight in A strains (mean 35,900) than B strains (mean 33,100). Small variations in the sizes of the F and G glycoproteins were not sufficient to suggest grouping on this basis.

Antigens, Viral↗
PubMed1995

Variation in the fusion glycoprotein gene of human respiratory syncytial virus subgroup A.

Six different genotypes (designated lineages SHL1-6) of human respiratory syncytial (RS) virus have been defined by partial nucleotide sequence analysis of the variable SH and the hypervariable G membrane protein genes, and by restriction fragment analysis of the conserved N protein gene of viruses isolated in south Birmingham. Viruses of very similar genotype appear to be present worldwide at the present time. We have determined the nucleotide sequences of the fusion protein genes of five viruses isolated in south Birmingham in the same year, but belonging to different lineages, and have compared them with the sequences of four subgroup A viruses isolated at earlier times from diverse localities. The sequence diversity of the F genes of these five viruses, as measured by nucleotide (94.5-98.5%) and inferred amino acid (97.0-99.3%) identifies, is comparable with that of the nine subgroup A viruses considered as a whole. No sequence changes occur in any of the sites of known epitopes. Comparison of the nine subgroup A sequences with the published sequences of a subgroup B strain and three bovine RS viruses confirms that the F protein sequences are most divergent in the F2 region.

Amino Acid Sequence↗
PubMedDate not supplied

Prevalence of antibodies against HEp-2 cell antigen in infants and children hospitalized with respiratory syncytial virus infection.

Infants with respiratory syncytial virus (RSV) infection were shown to have antibodies against HEp-2 cell antigen present in RSV-antigen preparation used for immunoblot analysis. The prevalence of anti HEp-2 cell antibodies was examined in infants hospitalized for RSV infection (n = 49, median age 121 days) compared to rotavirus infected children (n = 30, median age 114 days) and to healthy controls (n = 20, median age 150 days). The immunoblot analysis with RSV-infected and non-infected HEp-2 cells as antigen revealed the expected age-dependent low prevalence of G protein antibodies and clear seroconversion of N and P protein antibodies. HEp-2 antibody prevalence was higher in RSV antigen-positive infants (33/49) than in rotavirus antigen-positive (5/30) and RSV antigen-negative infants (4/20), respectively (p < 0.001). Anti HEp-2 antibodies were mostly directed against 47, 46, 33, 30 kD antigens. A multiple regression analysis found the following correlations (odds ratio; 95% confidence interval): 42 kD RSV antibodies (N protein) with pneumonia (7.58; 1.43-40), 94 kD RSV antibodies (G protein) with bronchiolitis (0.064; 0.006-0.686). This study shows repeated well-known features of humoral immunity in RSV infection. The data on anti HEp-2 antibodies point to a role for these pre-existing autoreactive antibodies in the pathogenesis of RSV infection.

Acute Disease↗