SYBR GREEN I-BASED REAL-TIME PCR REVEALED A HIGH INCIDENCE OF HUMAN BOCAVIRUS (HBOV) INFECTION IN THE PEDIATRIC POPULATION FROM PORTO ALEGRE, BRAZIL

Human bocavirus (hBoV) is a newly described respiratory virus detected in respiratory samples of patients with bronchiolitis. For this study we developed a real-time PCR (RT-PCR) assay using SYBR green I chemistry for detection of hBoV. Nasopharyngeal samples were collected over a one-year time period from 321 children admitted to a pediatric hospital in Porto Alegre, South of Brazil, presenting clinical symptoms and signs of bronchiolitis. Viral DNA was extracted from the respiratory samples using the QIAamp Ultrasens Viral Kit (QIAgen, Germany). First, a limited number of samples (~30) were analyzed by conventional PCR, as originally described by Allander T. et al., and those showing the expected amplification product were submitted to DNA sequencing to confirm the presence of hBoV. The positive samples, together with negative ones, were used to validate the real-time PCR assay. All 321 samples were analyzed by real-time PCR using a novel set of primers designed upon available genomic sequences of hBoV. RT-PCR was performed using the LightCycler platform (Roche) and a PCR Mix containing Sybr green I (Invitrogen, USA). Melting temperature (~86C) was used to confirm the presence of hBoV. A subset of the RT-PCR positive samples were further confirmed by direct DNA sequencing. Overall, 43 samples (13.4%) were positive for hBoV. According to the recently published papers, the prevalence of hBoV world-wide ranges from 3 to 15% and there are reports of mixed viral infection in some cases. In our study, for some patientes, hBoV was also found associated with other respiratory viruses, most notably with rhinovirus and metapneumovirus. The clinical significance of these mixed viral infections in unclear at the moment, but it may exacerbate the viral disease in some cases. We demonstrate here that patients from Porto Alegre, Brazil, have a high prevalence of hBoV in respiratory samples, and RT-PCR using SYBR green dye is a suitable detection method for this new viral agent.