Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>108 copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.

The objective of this study was to evaluate risk factors for persistent wheezing in a group of 2-4-year-old children after an index-wheezing episode in infancy. Eighty infants who had been seen at the Emergency Department for an episode of acute wheezing were followed for 2 yr in this prospective study. Caregivers completed a questionnaire, and children underwent clinical evaluation and skin prick testing 2 yr following the index-wheezing episode. Detection of respiratory viruses and analysis of exposure to major indoor allergens were carried out at enrollment. Immunoglobin E antibodies were measured at the beginning of the study and at the end of follow-up, using the CAP system. Logistic regression analysis was performed to identify factors associated with persistent wheezing. Seventy-three children (44 boys) completed the study. After 2 yr, 38 (52%) reported three or more wheezing episodes in the past 12 months (persistent wheezers). Independent risk factors for persistence of wheezing were allergic sensitization and exposure to cockroach allergen in the kitchen. Breast-feeding for at least 1 month was a protective factor. A strong association between allergic sensitization and persistence of wheezing was found in a group of very young children living in a subtropical area.

Human bocavirus (HBoV) was recently identified in respiratory samples from patients with acute respiratory infections and has been reported in different regions of the world. To the best of our knowledge, HBoV has never been reported in respiratory infections in Brazil. Nasopharyngeal aspirates were collected from patients aged <5 years hospitalized in 2005 with respiratory infections in Ribeirão Preto, southeast Brazil, and tested by polymerase chain reaction (PCR) for HBoV. HBoV-positive samples were further tested by PCR for human respiratory syncytial virus, human metapneumovirus, human coronaviruses 229E and OC43, human influenza viruses A and B, human parainfluenza viruses 1, 2 and 3, human rhinovirus and human adenovirus. HBoV was detected in 26/248 (10.5%) children of which 21 (81%) also tested positive for other respiratory viruses. Despite the high rates of co-infections, no significant differences were found between HBoV-positive patients with and without co-infections with regard to symptoms.

Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co-infections to the HRSV-disease have been based on the detection of HRSV by RT-PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co-detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable-HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT-PCR and virus isolation in cell culture. All samples with viable-HRSV were tested further by PCR for other respiratory viruses. HRSV-disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV-positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV-positive diseases were more severe than HRSV-negative ones, but there was no difference in disease severity between patients with viable-HRSV and those HRSV-positives by RT-PCR. Co-detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT-PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co-detection of other respiratory viruses was found in samples with viable-HRSV, but this was not associated with more severe HRSV infection.

Some risk factors for wheezing previously identified in temperate climates were present in a subtropical area, including respiratory syncytial virus infection in infants and allergy in children older than 2 years. Rhinovirus was not associated with wheezing and did not appear to be a trigger for asthma exacerbations.

Objective: Syncytia formation is the hallmark of the cytopathic effect caused by human respiratory syncytial virus (HRSV), which is the most important viral respiratory pathogen in children. This article reports methodological improvements in primary HRSV isolation and the importance of syncytia formation and mRNA levels of F protein for the progeny yield, using clinical isolates of HRSV. Methods: The A and B strains of HRSV were isolated in HEp-2 cell cultures from fresh and frozen nasopharyngeal aspirates. The formation of syncytia was evaluated using 2 different assays. Levels of F protein mRNA were quantified by real-time PCR while HRSV progeny titration was done by plaque assay. Results: HRSV was primarily isolated from 238 of 312 (90.7%) samples, and 13 of these (12 HRSV-A and 1 HRSV-B) were continuously passaged in vitro. The quantity and size of syncytia formed by 6 pure HRSV-A clinical isolates were different, as were the levels of F protein mRNA. Conclusion: There is a direct correlation of quantities of syncytia and inoculum size, but not with mRNA levels of HRSV-A F protein. Importantly, levels of F protein mRNA were directly related to progeny production.

Human adenovirus type 7 (HAdV-7) is an important cause of acute respiratory disease (ARD). Different genomic variants of HAdV-7 have been described, designated 7a-7l. In a previous study to investigate risk factors for ARD and wheezing, nasopharyngeal samples were collected from 90 ill children seeking medical attention in Ribeirã o Preto, Sã o Paulo, Brazil. HAdVs were identified in 31 samples and were characterized by serum neutralization and genome restriction analysis. Eleven HAdVs were identified as being HAdV-7, five of which were classified as being of genome type 7p (Gomen). Six other HAdV-7 isolates gave new restriction profiles with all enzymes used and were classified as being a new genomic variant, 7m. These isolates were further characterized by sequencing. The hexon and fiber genes of the 7m variant were nearly identical to the prototype, 7p. However, nucleotide sequences from the E3 cassette revealed a 1743 bp deletion affecting the 16. 1K, 19K, 20.1K and 20.5K ORFs.

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