The diagnosis of acute HBoV1 infection was proved by the presence of HBoV1-specific IgM and DNA in cell-free blood plasma as well as HBoV1 mRNA in PBMCs, whereas no other viruses or bacteria were found by PCR and culture, respectively. Acknowledgements The authors thank the childs legal guardian for allowing publication of the data and Professor Hsin-Fu Liu, Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan for gifting a HBoV1-containing plasmid. Abbreviations ARTIAcute respiratory tract infectioncDNAComplementary DNACRPC-reactive proteinEIAEnzyme immunoassayHBoV1Human bocavirus 1HBoV2Human bocavirus 2HBoV3Human bocavirus 3IgImmunoglobulinLRTILower respiratory tract infectionmRNAMessenger ribonucleic acidNPANasopharyngeal aspirateNPSNasopharyngeal swabPBMCsPeripheral blood mononuclear cellsPCRPolymerase chain reactionqPCRQuantitative polymerase chain reactionRNARibonucleic acidRSVRespiratory syncytial virusRTReverse transcriptaseVLPsVirus-like particlesWBCWhite blood cell Authors contributions IZ collected clinical samples, analyzed and interpreted data, and wrote the manuscript. with a Dextrorotation nimorazole phosphate ester history of rhinorrhea and cough for 6 days and fever for the last 2 days prior to admission, followed by severe respiratory distress and tracheal intubation. Human bocavirus 1 was the only respiratory virus detected by a qualitative multiplex polymerase chain reaction panel. For the diagnosis of acute human bocavirus 1 infection, both molecular and serological approaches were used. Human bocavirus 1 deoxyribonucleic acid (DNA) was detected simultaneously in nasopharyngeal aspirate, stool, and blood, as well as in the corresponding cell-free blood plasma by qualitative and quantitative polymerase chain reaction, revealing high DNA-copy numbers in nasopharyngeal aspirate and stool. Despite a low-load viremia, human bocavirus 1 messenger ribonucleic acid was found in the peripheral blood mononuclear cells. For detection of human bocavirus 1-specific antibodies, non-competitive immunoglobulin M and competitive immunoglobulin G enzyme immunoassays were used. The plasma was positive for both human bocavirus 1-specific immunoglobulin M and immunoglobulin G antibodies. Conclusions The presence of human bocavirus 1 genomic DNA in blood plasma and human bocavirus 1 messenger ribonucleic acid in peripheral blood mononuclear cells together with human bocavirus 1-specific immunoglobulin M are markers of acute human bocavirus 1 infection that may cause life-threatening acute bronchiolitis. DNA, as described [20]. An HBoV1-containing plasmid was used as a positive control in PCR. All these samples were HBoV1 DNA positive. Upon re-examination by quantitative PCR (qPCR) (Human bocavirus genomes, Standard kit, Genesig, Primerdesign Ltd., UK), the copy numbers in NPA and stool were high, 5.7??105 per g DNA in NPA and 1.4??108 per g DNA in stool. The viral load in blood was 21 copies/g DNA, but in cell-free blood plasma the viral load was under detection level. To prove that the HBoV1 infection was actively ongoing, HBoV1 transcription in PBMCs was applied. Total ribonucleic acid (RNA) was extracted from PBMCs using TRI Reagent? solution according to the manufacturers instructions (Thermo Fisher Scientific, USA). The extracted RNA was quantified spectrophotometrically and analyzed by electrophoresis in a 1% agarose gel. RNA was treated with DNase (TURBO DNA-free? Kit, Thermo Fisher Scientific, USA) before the synthesis of complementary DNA (cDNA) by the reverse transcriptase (RT) using RevertAid? First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The gene sequence was detected by PCR to assess the quality of synthesized Dextrorotation nimorazole phosphate ester cDNA (Fig.?2). Open in a separate window Fig. 2 Electrophoretic visualization of amplification products in a 1% agarose gel after polymerase chain reaction targeting gene sequence HBoV1-specific Dextrorotation nimorazole phosphate ester cDNA was detected by PCR Rabbit polyclonal to FANK1 targeting the HBoV1 gene as described by Sloots gene. em Legend 1 /em : em 1 /em . pUC19 DNA/MspI (HpaII) marker; em 2 /em . negative control (molecular biology grade H2O); em 3 /em . complementary DNA sample synthesized from DNase treated ribonucleic acid; em 4 /em . DNase treated ribonucleic acid sample without reverse transcriptase step. em Legend 2 /em : em 1 /em . pUC19 DNA/MspI (HpaII) marker; em 2 /em . DNase treated ribonucleic acid sample without reverse transcriptase step; em 3 /em . complementary DNA sample synthesized from DNase treated ribonucleic acid; em 4 /em . negative control (molecular biology grade H2O); em 5 /em ., em 6 /em . positive control (human bocavirus 1 plasmid); em 7 /em . GeneRuler 100?bp DNA Ladder Biotinylated virus-like particles (VLPs) of the recombinant major capsid protein VP3 were used as antigen in enzyme immunoassays (EIAs) for detection of HBoV1-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) in our patients plasma sample [23, 24]. For removal of possible cross-reacting heterologous human bocavirus 2 (HBoV2) and human bocavirus 3 (HBoV3) IgG, non-biotinylated VLPs in competition assays were.