Detection of DNA virus groups in 20

a<p>CR ELISA is genus specific for orthopoxviruses but not ECTV species specific.</p>b<p>this quantity cannot be calculated.</p>c<p>ECTV.</p>d<p>MAdV.</p>e<p>MPV, MVM, H-1 PV, RPV, KRV and RMV.</p>f<p>MPyV and K-virus.</p

A Bead-Based Multiplex Assay for the Detection of DNA Viruses Infecting Laboratory Rodents

<div><p>The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals’ physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.</p></div

Detection limits (DL) of rDVF for rodent DNA viruses.

a<p>determined in duplicates.</p

DNA viruses in pet shop rodents.

<p>Prevalence (y-axis) of DNA viruses (x-axis, categories) shown in mice (striped bars) and in rats (white bars). MPV1–5 is summarised as MPV, MVMi and MVMp as MVM and MAdV-1 and -2 as MAdV. RRHV summarises <i>Rattus rattus</i> rhadinovirus and <i>Rattus norvegicus</i> rhadinovirus.</p

Specificity of rDVF.

a<p>specific MFI value.</p>b<p>murine DNA.</p>c<p>expected cross-reaction due to high sequence homology.</p>d<p>rat DNA.</p

Number of HPV positive samples.

<p>Number of HPV genotypes detected in oral rinse and gargle samples of the 500 participants. One participant had a co-infection, HPV16 and HPV81. Black bars, high-risk HPV; grey bars, low-risk HPV. HPV human papillomavirus.</p

History of Pap smear testing and oral HPV status.

<p>History of Pap smear testing and oral HPV status.</p

Number of heterosexual sex partners stratified by oral HPV status.

<p>Number of heterosexual sex partners stratified by oral HPV status.</p

Expression and secretion of IL-1β in human primary keratinocytes and HPV-positive cell lines.

<p>A) Quantification of secreted IL-1β (expressed as pg/ml) by ELISA in human primary keratinocytes (PK), keratinocytes immortalized by individual oncogenes (E6, E7, E6/E7) and HPV16/18-positive cervical carcinoma cell lines (SiHa, CaSki, HeLa, respectively) 24 h after infection with a recombinant GFP-expressing adenovirus 5 (Ad) in comparison to uninfected cells. B) Quantification of basal intracellular IL-1β levels by ELISA. C) Western blot analysis of pro-IL-1β in human primary keratinocytes (PK) and HPV-positive cells. Actin was used as a loading control. D) qPCR analysis of IL-1β cDNA obtained from PK and HPV-positive cells (Ordinate: expressed as fold changes using the average IL-1β steady state level of 3 different primary human keratinocyte preparations PK cells as a reference which was arbitrarily set as 1. The graphs in A, B and D represent the mean values (± SEM) of three independent experiments. ANOVA ***p<0.05.</p

Analysis of IL-1β expression in normal and HPV16-positive cervical tissues.

<p>A) Immunohistochemical analysis of IL-1β expression in normal epithelium and HPV-positive lesions differing in their progression grades CIN I to CIN III and cervical tumors; scale bars represent 25 µm. B) qPCR analysis of IL-1β cDNA derived from samples negative for intraepithelial lesion and malignancy (NT), different HPV-positive lesions (CIN I, II, III) and cervical tumors (CxCa); ordinate: expression as fold changes using SiHa cells as reference which was arbitrarily set as 1. The pictures in A are a representative example of 25 biopsies analyzed from normal tissue (n = 5) and HPV-positive lesions of different donors (n = 25). The box-and-whisker blot in B represents the mean values from three to five samples for each group depicted in the graph (± SEM). ANOVA *p<0.01.</p