This figure shows selected representative fluorescence microscopy images from HBoV DNA positive tumor samples and control cells.

<p><b>a</b>. Rows 1-3 show tissues stained with HBoV-specific probes and DAPI; rows 4-6 show positive and negative controls. LEH and REH correspond to the left end (5’-end) and the right end (3’-end) of the viral genome or the GAPDH gene. Human cells transfected with plasmids with or without human bocavirus genomes as well as mock transfected cells were used as controls. Row 7 shows HepG2 cells stained with probes specific for terminal sequences of the GAPDH gene. <b>b</b>. Enlargement of the merged image of HepG2 cell double stained with probes specific for the human GAPDH gene. This figure shows that the distance between the two different probes at the 5’-end and the 3’-end is large enough to result in separate signals (split signal). <b>c</b>. Merged images of HBoV DNA positive tissues, including a negative control tissue.</p

NF-κB expression in the lung after infection.

<p>Amount of NF-κB in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c-mice were infected with 2×10⁷ geq RSV, or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed, homogenised. The amount of NF-κB was determined by ELISA. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05). # signifcantly different to cell culture infected animals (p<0.05). + significantly different to hMPV and hMPV/RSV-infected animals (p<0.05).</p

TNF-α expression in the lung after infection.

<p>Amount of TNF-α in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed and homogenised. The amount of TNF-α was determined by ELISA. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05). # signifcantly different to cell culture infected animals (p<0.05).</p

Soluble Collagen Expression in the infected lung.

<p>Amount of soluble collagens in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV, or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed and homogenised. The amount of soluble collagens was determined by a collagen assay. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05).</p

Viral load of lungs after infection.

<p>Viral load of lungs obtained from 4–6 weeks and 19 months old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed, homogenised and viral RNA was extracted. The number of genome equivalents was estimated by qRT-PCR (n = 3–5, values are shown as mean ± standard deviation).</p

Weight change of infected animals.

<p>Weight change of 4–6 weeks and 19 month old BALB/c-mice before and after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Animals were weighed daily. All values were set in relation to the weight of the animals on day 1. The red line marks the day of infection (n = 5, values are shown as mean).</p

Daily food consumption of infected animals.

<p>Daily food-consumption of 4–6 weeks (A) and 19 month (B) old BALB/c mice before and after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. The food consumption was recorded at the beginning of the experiment, on the day of inoculation and at the end of the experiment. Values were standardized referring to the values before infection (n = 3–5, values are shown as mean ± standard deviation). + significanty different to the corresponding value before infection (b.i.) (p<0.05).</p

Alignment of head and tail sequences of several published HBoV isolates.

<p>Most isolates differ in the length of the published sequences. Red boxes indicate primer binding sites of the head and tail primers used in this study, respectively. Nucleotide numbering is according to the longest isolate sequences used in the alignment.</p

Approach for deciüphering the hairpin like structure of human bocavirus.

<p><b>a: </b><b><i>Putative hairpin structures of human bocavirus.</i></b> In order to test for the self priming capability of the HBoV genome during genome replication different polymerases were added to HBoV DNA isolated from clinical samples. Both, positive strand and negative strand containing isolates were used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019457#pone.0019457-Bohmer1" target="_blank">[23]</a>. Following self-priming elongation the DNA was denaturized and PCR was performed with a single primer. Surprisingly in none of the tested isolates a PCR product was observed, leading to the conclusion that no self priming occurred. <b>b: </b><b><i>HBoV end terminus PCR with primer Nrul1 and Ssp1, respectively.</i></b> HBoV genome preparations were incubated with T7 polymerase and subject to subsequent PCR with primers Boca_end_NruI-1_neu and Boca_end_SspI-1_neu, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019457#pone-0019457-g001" target="_blank">figure 1a</a>. <b>c: </b><b><i>HBoV end terminus PCR with primer Nrul2 and Ssp2, respectively.</i></b> HBoV genome preparations were incubated with Klenow polymerase and subject to subsequent PCR with primers NruI-2 and SspI-2, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019457#pone-0019457-g001" target="_blank">figure 1a</a>. <b>d: </b><b><i>HBoV end terminus PCR after circularisation of the genome with primer Nrul2 and Ssp2.</i></b> HBoV genome preparations were incubated with T4 RNA ligase (self-ligation of single strand genomes and subject to subsequent PCR with primers Nru2 and Ssp2, respectively. Theoretically, head-to-tail sequences should have been amplified provided the terminal regions of the genome allow self-ligation and are not masked by secondary structures resistant to self-ligation. <b>e: </b><b><i>HBoV PCR with primer set Boca_end_SspI-1_neu and Boca_end_NruI-1_neu.</i></b> This PCR approach was performed with primer in the outmost terminal region of the HBoV genome DQ000496. The PCR reaction should have amplified head-to-tail, tail-to-tail, or head-to-head structures, provided the target sequence is present in the clinical isolates. Unfortunately the primers did not bind to a terminal region that is know among all so far published isolates, thus it remains unclear whether primer binding was sufficient.</p

Overview on primers used in this study.

<p>The table summarizes sequences, genome locations, and names of primers used in the study. Primer were used in combinations as indicated in the text and for detection of intermediate DNA sequences as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019457#pone-0019457-t002" target="_blank">table 2</a>; *GenBank entry DQ000496 covers the first published HBoV isolate and presents the current reference sequence. (+) = forward; (−) = reverse.</p