The author engaging in the process of scientific discovery.

<p>The author engaging in the process of scientific discovery.</p

Schematic representation of the HBoV3 episome, results of inverse PCR and sequence alignment of HBoV genomic termini.

<p>(A) Describes the genomic organization of the HBoV3 episome and location/orientation of PCR primers used for screening samples and inverse PCR. (B) Shows results of inverse PCR assay for three samples (B-1 to B-3) that were positive for HBoV3 screening PCR with molecular weight (MW) marker and negative (Neg) reagent PCR control. (C) Shows alignment of previously known genomic termini of HBoV3 and HBoV1 linear genomes with the non-coding region of HBoV3 episome. Unique sequence identified in this study is shown as connecting the 3′ and 5′ termini (nucleotide with no background and “-” represent sequence that remained elusive in previous studies).</p

Secondary structure prediction of the sequences immediately downstream of the structural gene of different HBoV species (upper-HBoV1, middle-HBoV2 and lower-HBoV3-E1).

<p>The stems and arms are numbered for comparison, and the termination codon of the VP gene is marked by a solid line. The rabbit-ear structure (structure 3 and 4) present in all 3 HBoV species is comparable to similar conserved structures of left-hand side termini reported for animal bocaviruses (MVC and BPV) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021362#pone.0021362-Sun1" target="_blank">[44]</a>.</p

Secondary structure prediction and sequences for the non-coding terminal region of HBoV3-E1 (top) and MVC (bottom).

<p>The 3 major bocavirus genes are shown in shaded boxes as NS, NP and VP. The NS gene protein initiation codon and VP genes stop codon are underlined. The 2 stable and long palindromic sequences are shown as hairpin 1 and 2. Cluster of DNA stems and loops immediately upstream of the N-terminus of the NS gene are shown as 5′ terminal structures. Based on the Genbank data, the start and end of the HBoV and MVC linear genomes are shown with arrows.</p

Comparative phylogenetic analysis of HBoV3-E1 and HBoV2-IB1 (filled rectangles).

<p>Nucleotide alignments were generated using the complete coding sequence of the NS, NP and VP genes. Names of sequences used for analysis are shown as Genbank accession numbers followed by the names of HBoV species and strains. The tree was constructed with the maximum likelihood method using a GTR+G substitution model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021362#pone.0021362-Tamura1" target="_blank">[38]</a>. Bootstrap replicates (>70%) are shown above the branches and distances (>0.02) are shown below the braches.</p

Prevalence of HMOAstV-C antibodies with childhood age.

<p>A total of 103 child serum samples were analyzed by LIPS including from the following age brackets: 0–6 months (n = 22), 6–12 month (n = 15), 1–2 year (n = 22), 2–5 year (n = 22), and 5–10 year olds (n = 22). Raw LU values are shown without subtracting background binding to protein A/G beads. The dashed line represents the diagnostic cut-off value derived from the mean plus 3SD of replica buffer blank samples. The fraction and percent seropositive for HMOAstV-C are shown above each group.</p

Comparison of the C-terminal capsid fragment of HMOAstV-C with related viruses.

<p>From BLASTP analysis, the highest homology of the HMOAstV-C capsid fragment was with HMOAstV-B (68% identity) and HMOAstV-A (30% identity) astroviruses. Identical amino acid residues with HMOAstV-C are shaded.</p

LIPS detection of antibodies to a C-terminal capsid fragment of HMOAstV-C.

<p>Antibodies to the N- and C-terminal capsid protein of HMOAstV-C and the C-terminal capsid protein of HMOAstV-CA capsid protein fragments were analyzed in 45 adult serum samples. Each symbol represents individual serum samples tested with each protein fragments and LU values were adjusted by subtracting background binding to protein A/G beads. The short solid line represents the mean antibody titer for each group.</p

Comparison of pairwise nucleotide and amino acid (bold typeface) distances (p-distance) of all four genes between GBoV1 and HBoV species confirms GBoV1 as prototype of a new bocavirus species.

<p>Comparison of pairwise nucleotide and amino acid (bold typeface) distances (p-distance) of all four genes between GBoV1 and HBoV species confirms GBoV1 as prototype of a new bocavirus species.</p

Phylogenetic analysis of GBoV1.

<p>(A) Full length structural protein (VP1/2) sequences of all HBoV and animal bocavirus strains available in GenBank were used to determine phylogeny of GBoV1 by neighbor-joining analysis of pairwise distances between translated amino acid sequences. Bootstrap re-sampling was used to determine robustness of individual clades (values above 70% shown above the branches). (B) The 4 major open reading frames of GBoV1 were analyzed using maximum likelihood composition analysis method (MEGA4.1) comparing pairwise distances of translated sequences of representative variants (reference sequence) of all four HBoV species. Accession numbers of sequences used precedes the name of corresponding bocavirus species.</p