Texas Pediatric Society Electronic Poster Contest Determining Persistence of Bocavirus DNA in the Respiratory Tract of Children by Pyrosequencing J Castro Wagnera; R Pylesac; A Millera; J Nokso-Koivistoa; M Loeffelholzb; T Chonmaitreeab Departments of aPediatrics, bPathology, cMicrobiology and Immunology. University of Texas Medical Branch. Galveston, Texas. USA Abstract Human bocavirus type 1 (HBoV1) is a pathogen of the respiratory tract. Presence of HBoV1 in nasopharyngeal secretions of asymptomatic children is not uncommon. Because of prolonged presence of the virus after acute infection, detection of HBoV1-DNA in the clinical specimen doesn’t always indicate newly acquired or active infection. We used DNA sequencing to differentiate new from persistent infection in children (6 to 36 mos. of age) in a prospective, longitudinal study. Each child was followed for one year for upper respiratory tract infection (URI) and acute otitis media (AOM), 94 children tested positive for HBoV DNA during 172 URI episodes. Archived HBoV DNA from these samples were subjected to polymerase chain reaction (PCR) followed by targeted DNA sequencing through pyrosequencing of genes: NP-1 for speciation and VP-1, VP-2 for subtyping. Infections with HBoV were mainly found to be prolonged (up to 181+d) in the respiratory tract continuously or intermittently. Recurrence after 8-9 months was identified suggesting possible persistence and reactivation. Infections with 2 different HBoV strains in a child within one-year period were uncommon. Newly acquired infection with HBoV1 (without co-infection) was commonly complicated by AOM. Table 2. Human bocavirus type 1 (HBoV1) strains identified: (n=108 fully sequenced specimens). Table 3. HBoV1 infection patterns in children with multiple URI episodes. Introduction Human bocavirus 1 (HBoV1), member of the Parvoviridae family, has a global impact, with a prevalence of up to 33% in young children with respiratory tract infections.(1-3,8) HBoV1 has been associated with both upper (URI), lower respiratory tract infections (LRI) and acute otitis media (AOM); affecting predominantly children 6-24 months old. (1-7) Prolonged HBoV1 presence in asymptomatic children for up to 6 months has been previously described, (6,7) and latent infection with subsequent reactivation has also been hypothesized.(3) Therefore, detection of HBoV1 in a clinical specimen doesn’t always indicate new HBoV1 infection.   Our aim was to characterize HBoV1 nucleic acid sequences detected in the respiratory tract of children to differentiate a newly acquired infection from prolong presence and to determine the significance of HBoV1-URI in AOM development. Figure 2. Examples of multiple HBoV1 cases. Each specimen collected during symptomatic URI is represented by a vertical hash mark. HBoV1 strains are displayed in grey-boxes. HBoV1 co-infected with other respiratory virus (es) indicated with “+other”. Cases: 1: newly acquired infection with clearance <30 days; 2: prolonged presence of >30 days; 3: intermittently presence with negative samples in between; 4: recurrence or reactivation (323 and 244 days); 5: successive HBoV1 detection with insufficient follow up; 6: different HBoV1 strains in <1 year. Abbreviations: Neg: HBoV PCR (-). L/T: LWK/TW strain; L/BJ: LWK/BJ strain; B/BJ: Bonn/BJ strain; L/BJ: LWK/BJ strain. Other or +other: different respiratory viruses (i.e.: rhinovirus, adenovirus, enterovirus, echovirus, RSV.) Description of Study Archived specimens from a prospective study (January 2003-March 2007) of healthy children 6-36 months old, followed for 1 year for upper respiratory tract infection (URI) and AOM. Nasopharyngeal secretions (NPSs) were collected during URI episodes (presence of nasal congestion, rhinorrhea, cough, and/or sore throat with or without fever). DNA from 172 HBoV1(+) specimens were subjected to PCR for optimization, followed by targeted DNA pyrosequencing for regions within NP1, VP1 and VP2 genes for identification and comparison. AOM incidence was compared to HBoV1 newly acquired infection. Results Of 172 HBoV(+) specimens, HBoV DNA successfully sequenced in 128 (74%) samples (from 70 children); all were HBoV type 1. Four main HBoV1 strains identified: LWK/TW (63%), LWK/BJ (20%), Bonn/BJ (16%) and LWK/KU3 (1%). Of 70 children, 29 had only one HBoV1 detection; 41 children had 2 or more HBoV(+) samples. 17 of 70, failed sequencing; 53 children had 108 adequate HBoV1 sequencing results. Of 53 children, 24 had single HBoV1 sequence each. A total of 29 children with 190 NPA samples yielded 84 HBoV1 sequencing results available for comparison (2-4 sequences each). Findings as follows: Table 4. Comparison of HBoV1 infection alone (without co-infection) in URI episodes with and without AOM. 1 New infection: HBoV1 (+)-URI episode preceded by negative HBoV1 sample and no recent HBoV1 infection. Initial URI HBoV1 (+) since enrollment. 2 Persistent infection: sample preceded by HBoV1 (+) URI 3 Possible persistent infection: sample preceded by recent URI with same HBoV1 (+) sequence although HBoV1 (-) sample in between. Figure 1. Flow diagram for subjects and specimens included in the study. Conclusions HBoV1 infections often result in prolonged presence (up to 181d) of DNA in the respiratory tract continuously or intermittently. Recurrence after 8-9 mos. suggests the possibility of persistence and reactivation. Infections with 2 different HBoV1 strains in a child within one-year period are uncommon. Newly acquired HBoV1-URI (without co-infection) is often complicated by AOM. 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Distinctive clinical features of human bocavirus in children younger than 2 years. Eur J Pediatr 2010; 169:1087–92. Martin ET, Fairchok MP, Kuypers J, et al. Frequent and prolonged shedding of bocavirus in young children attending daycare. J Infect Dis 2010; 201:1625–32. Lehtoranta L, Söderlund-Venermo M, Nokso-Koivisto J, et al. Human bocavirus in the nasopharynx of otitis-prone children. Int J Pediatr Otorhinolaryngol 2012; 76:206–11. Allander T. Human bocavirus. J Clin Virol. 2008 Jan; 41(1): 29-33. Texas Pediatric Society Electronic Poster Contest