Detection of head-to-tail DNA sequences of human bocavirus in clinical samples

Abstract

Parvoviruses are single stranded DNA viruses that replicate in a so called "rolling-hairpin" mechanism, a variant of the rolling circle replication known for bacteriophages like φX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus.

Figure 1. Approach for deciüphering the hairpin like structure of human bocavirus.

a: Putative hairpin structures of human bocavirus. 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 . 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: HBoV end terminus PCR with primer Nrul1 and Ssp1, respectively. 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 figure 1a. c: HBoV end terminus PCR with primer Nrul2 and Ssp2, respectively. 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 figure 1a. d: HBoV end terminus PCR after circularisation of the genome with primer Nrul2 and Ssp2. 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. e: HBoV PCR with primer set Boca_end_SspI-1_neu and Boca_end_NruI-1_neu. 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.

Figure 2. Theoretical structure of concatemeric sequences that could occur during HBoV replication.

The boxes indicate the primers that were used for the amplification of the putative concatemeric structures. The broken lines indicate the linking sequence that is dependent on the length of the unknown terminal sequences.

Figure 3. Alignment of head and tail sequences of several published HBoV isolates.

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.

Figure 4. Agarose gel elctrophoreses of PCRs with all possible combinations of head- and tail-primer pairs.

Two representative gel pictures are shown. All bands were gel extracted and subject to sequencing and cloning and sequencing from both directions. Arrows indicate the band that contained the HBoV head-to-tail sequences.

Figure 5. Human bocavirus forma head-to-tail sequences.

a. Agarose gel electrophoreses of head-to-tail PCR from different clinical HBoV-isolates. Arrows indicate that the corresponding band was gel-extracted and sequenced. Sequences are aligned in figure 3b. b. Alignment of Head-to-Tail PCR product from different isolates and cell culture of HBoV. All PCR products contained a sequence from the tail of several published clinical isolates that except single point mutations perfectly matches with the prototype sequence of the “Allander strain” DQ000496 (Tail), nt5254–nt5319. This region is followed by a so far unknown sequences, here referred to as linking sequence, which in turn is followed by head sequences conserved in several strains, again perfectly matching to the “Allander strain” DQ000496 (Head), nt3–nt219. The sequence of the PCR amplificates is highly compatible with a classical rolling circle replication (fig. 3c).