Relating plaque morphology to respiratory syncytial virus subgroup, viral load, and disease severity in children.

BACKGROUND: Viral culture plaque morphology in human cell lines are markers for growth capability and cytopathic effect, and have been used to assess viral fitness and select preattenuation candidates for live viral vaccines. We classified respiratory syncytial virus (RSV) plaque morphology and analyzed the relationship between plaque morphology as compared to subgroup, viral load and clinical severity of infection in infants and children. METHODS: We obtained respiratory secretions from 149 RSV-infected children. Plaque morphology and viral load was assessed within the first culture passage in HEp-2 cells. Viral load was measured by polymerase chain reaction (PCR), as was RSV subgroup. Disease severity was determined by hospitalization, length of stay, intensive care requirement, and respiratory failure. RESULTS: Plaque morphology varied between individual subjects; however, similar results were observed among viruses collected from upper and lower respiratory tracts of the same subject. Significant differences in plaque morphology were observed between RSV subgroups. No correlations were found among plaque morphology and viral load. Plaque morphology did not correlate with disease severity. CONCLUSION: Plaque morphology measures parameters that are viral-specific and independent of the human host. Morphologies vary between patients and are related to RSV subgroup. In HEp-2 cells, RSV plaque morphology appears unrelated to disease severity in RSV-infected children.

Respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children.

BACKGROUND: Respiratory syncytial virus (RSV) disease severity was thought to be a result of host immunopathology but alternatively may be driven by high-level viral replication. The relationships between RSV load, viral clearance dynamics, and disease severity have not been carefully evaluated. METHODS: Previously healthy RSV-infected children <2 years old were recruited. RSV load was measured in respiratory secretions by fresh quantitative culture over 3 hospital days. Measures of disease severity were hospital admission, duration of hospitalization, requirement for intensive care, and respiratory failure. RESULTS: Multivariate logistic regression models revealed independent predictors of increased duration of hospitalization: male sex, lower weight, and higher viral load on any day. Viral loads at day 3 were more significantly associated with requirement for intensive care and respiratory failure than were viral loads at earlier time points. Faster RSV clearance was independently associated with shorter hospitalization. DISCUSSION: These observations challenge the immunopathology-based pathogenesis paradigm. They also have major therapeutic implications, suggesting that application of antiviral agents early in the disease course, even at a time when viral replication is at its highest, might improve subsequent morbidity by significantly lowering viral load and direct viral cytopathic effects, and aborting the potential downstream immunopathology.

Viral Specific Factors Contribute to Clinical Respiratory Syncytial Virus Disease Severity Differences in Infants.

BACKGROUND: There is a wide range of severity of respiratory syncytial viral (RSV) disease in previously healthy infants. Host factors have been well demonstrated to contribute to disease severity differences. However the possibility of disease severity differences being produced by factors intrinsic to the virus itself has rarely been studied. METHODS: Low-passage isolates of RSV collected prospectively from infants with different degrees of RSV disease severity were evaluated in vitro, holding host factors constant, so as to assess whether isolates induced phenotypically different cytokine/chemokine concentrations in a human lung epithelial cell line. Sixty-seven RSV isolates from previously healthy infants (38 hospitalized for acute RSV infection (severe disease) and 29 never requiring hospitalization (mild disease)) were inoculated into A549, lung epithelial cells at precisely controlled, low multiplicity of infection to mimic natural infection. Cultures were evaluated at 48 hours, 60 hours, and 72 hours to evaluate area under the curve (AUC) cytokine/chemokine induction. RESULTS: Cells infected with isolates from severely ill infants produced higher mean concentrations of all cytokine/chemokines tested (IL-1α, IL-6, IL-8 and RANTES) at all-time points tested. RSV isolates collected from infants with severe disease induced significantly higher AUCIL-8 and AUCRANTES secretion in infected cultures than mild disease isolates (p=0.028 and p=0.019 respectively). IL-8 and RANTES concentrations were 4 times higher at 48 hours for these severely ill infant isolates. Additionally, 38 isolates were evaluated at all-time points for quantity of virus. RSV concentration significantly correlated with both IL-8 and RANTES at all-time points. Neither cytokine/chemokine concentrations nor RSV concentrations were associated with RSV subgroup. DISCUSSION: Infants' RSV disease severity differences may be due in part to intrinsic viral strain-specific characteristics.

Opportunities for early therapy of respiratory syncytial virus (RSV) infection: what happens before hospitalization.

RSV loads of infants are already declining near the time of hospitalization. For optimal effect, antiviral and other RSV therapeutics therefore may need to be applied before this occurs. 134 RSV-hospitalized infants were studied to determine the timing of events and their healthcare seeking behavior prior to hospitalization. Sixty-two percent of infants had contact with a health care professional > or = 1 day prior to hospitalization and 46% had such a contact on > or = 2 different days prior to hospitalization. The mean (S.D.) duration of RSV symptoms prior to hospitalization was 3.9 +/- 2.3 days and prior to first health care contact was 2.6 +/- 1.8 days. RSV-hospitalized infants routinely have contact with health care professionals at significantly early time points in their illness. These contacts are potential opportunities for initiation of early anti-RSV therapy.

Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children. Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 153:1. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r2 = 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 +/- standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r2 = 0.158; P < 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P < 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.

Respiratory syncytial virus (RSV) loads in premature infants with and without prophylactic RSV fusion protein monoclonal antibody.

It has not been determined whether respiratory syncytial virus (RSV) fusion protein monoclonal antibody (MAb) (palivizumab) reduces infection or simply ameliorates disease. In a prospective observational study, 27 hospitalized premature infants with RSV who were not receiving MAb were compared with 10 such patients who were receiving MAb. Mean (SEM) RSV loads in the MAb and non-MAb groups were 3.36 (0.59) versus 4.89 (0.27) logPFU/ml (P=.01). Prophylactic palivizumab reduces nasal RSV in premature hospitalized infants.