Human Bocavirus in Hospitalized Children, South Africa

To the Editor: In recent years, several novel respiratory viruses have been identified. These include human metapneumovirus (HMPV) (1), severe acute respiratory syndrome– associated coronavirus (2), human coronavirus (HCoV) NL63 (3,4), HCoV HKU1 (5), and, most recently, human bocavirus (HBoV) (6). The latter belongs to the Parvoviridae family and is most closely related to bovine parvovirus and canine minute virus (CnMV), which are members of the genus Bocavirus (6). Parvovirus B19 and HBoV are the only 2 par-voviruses known to be pathogenic to humans, but the relevance of HBoV infection in the clinical setting is not known. In this retrospective study, 341 nasopharyngeal and bronchoalveolar lavage samples were taken from children (age 2 days–12 years) hospitalized with respiratory tract infections in 2004 in the Red Cross War Memorial Children's Hospital, Cape Town, South Africa. Samples were originally screened by using an indirect immunofluorescence assay) for common respiratory viruses, including respiratory syncytial virus; influenza virus A and B; parainfluenza viruses 1, 2, and 3; adenovirus; and cytomegalovirus. Subsequently, HMPV and HCoV NL63 were detected by using reverse transcription–PCR (1,3). Samples were also screened for HBoV DNA. DNA was extracted by using the QIAamp DNA blood mini kit according to the manufacturer's instructions region of the NP-1 gene and the 3′ portion of the VP1/2 capsid gene of HBoV was performed. Briefly, 10 µL DNA was added to a 50-µL PCR mix containing 2 IU Supertherm poly-merase (JMR Holdings, Kent, UK), 1.5 mmol/L MgCl 2 , 200 µmol/L each dNTP, and 0.2 µmol/L primers NP-1 s1 (5′-TAACTGCTCCAGCAAGTC-CTCCA) and NP-1 as1 (5′-GGA-AGCTCTGTGTTGACTGAAT). To improve sensitivity, a second semi-nested reaction with 2.5 µL outer product and NP-1 as1 primer and NP-1 s2 (5′-CTCACCTGCGAGCTCTG-TAAGTA) primer was performed at an annealing temperature of 55°C. Negative controls were used, and appropriate measures were taken to prevent contamination (7). Samples with an NP-1–specific PCR product of 368 bp were confirmed by amplifying a 980-bp product of the VP1/2 capsid gene in a similar seminested PCR amplification protocol (primers VP s1 5′-GCACTTCTGTATCAGAT-GCCTT, VP as1 5′-CGTGGTATG-TAGGCGTGTAG, and VP s2 5′-C T TA G A A C T G G T G A G A G-CACTG). A selection of the inner VP1/2 amplicons obtained from samples taken over the year were sequenced directly and aligned in ClustalX, and a phylogenetic tree was constructed with the Kimura 2-parameter neighbor-joining method with 1,000 bootstrap resamplings. Comparative sequences were obtained from GenBank and included …