A method for the infectivity discrimination of enveloped DNA viruses using palladium compounds pre-treatment followed by real-time PCR.

Cultivation-based assays represent the gold standard for the assessment of virus infectivity; however, they are time-consuming and not suitable for every virus type. Pre-treatment with platinum (Pt) compounds followed by real-time PCR has been shown to discriminate between infectious and non-infectious RNA viruses. This study examined the effect of Pt and palladium (Pd) compounds on enveloped DNA viruses, paying attention to two significant pathogens of livestock - bovine herpesvirus-1 (BoHV-1) and African swine fever virus (ASFV). Native or heat-treated BoHV-1 suspension was incubated with the spectrum of Pt/Pd compounds. Bis(benzonitrile)palladium(II) dichloride (BB-PdCl 2) and dichloro(1,5-cyclooctadiene) palladium(II) (PdCl 2-COD) produced the highest differences found between native and heat- -treated viruses. Optimized pre-treatment conditions (1 mM of Pd compound, 15 min, 4°C) were applied on both virus genera and the heat inactivation profiles were assessed. A significant decrease in the detected quantity of BoHV-1 DNA and ASFV DNA after heat-treatment (60°C and 95°C) and consequent incubation with Pd compounds was observed. BB-PdCl 2 and PdCl 2-COD could help to distinguish between infectious and non-infectious enveloped DNA viruses such as BoHV-1 or ASFV.

Human rotavirus A detection: Comparison of enzymatic immunoassay and rapid chromatographic test with two quantitative RT-PCR assays.

OBJECTIVE: The aim of this study was to compare results of two commercially available kits used for routine detection of Rotavirus A in human stool samples with results of commercial quantitative reverse-transcription PCR (RT-qPCR) test and in-house RT-qPCR. MATERIAL AND METHODS: In total, 749 stool samples were screen-ed with the use of four different methods. The samples were collected from four diagnostic laboratories from March 2016 to June 2017. Diagnose of gastrointestinal disorders was stated in one third of tested patients, the rest of samples was collected from patients with other primary diagnose. The samples were tested with the enzymatic immunoassay (EIA) (RIDASCREEN® Rotavirus) and with rapid diagnostic immunochromatographic test (RDT) (IMMUNOQUICK® No-Rot-Adeno). As a reference method a commercial RT-qPCR test was used (Primerdesign Genesig® Kit) and it was compared with in-house RT-qPCR test prepared in our laboratory. The samples which in the reference RT-qPCR gave positive signal of reaction in cycle 28 or higher (Ct 28) were assessed as negatives in order to include only samples with some clinical relevance into sensitivity determination. RESULTS: Diagnostic sensitivity was assessed as 84.2% for EIA and 82.5% for RDT. The specificity of those tests was calculated as 97.8% for EIA and 96.4% for RDT. The performance of both diagnostic tests describing their positive predictive value was determined to be 87.3% for EIA and 80.3% for RDT. Negative predictive value was calculated to be 97.2% for EIA and 96.8% for RDT. Proportion of RVA-positive samples determined with the reference RT-qPCR test with our own cut-off level was 15.2% (n=114). Comparisons of the in-house and reference RT-qPCR tests showed very good agreement of results. The sensitivity of the in-house test was 100% and its specificity 99.7%. CONCLUSIONS: RT-qPCR is more sensitive for surveillance of rotavirus gastroenteritis than routinely used EIA or RDT methods. The specificity of both evaluated tests was very high. However, EIA was in all performance parameters assessed better than RDT.

[The possibilities of zoonotic transmission of rotaviruses]. / Moznosti zoonotického prenosu rotavirových infekcí.

Rotaviruses are significant enteric pathogens of humans and animals. Rotavirus-associated mortality is still high in children, especially in developing countries. Rotaviruses also account for important economic losses due to gastrointestinal disease of livestock animals, notably of young cattle and pigs. Increasing numbers of different rotavirus strains stress the necessity of their uniform nomenclature and detailed classification with the use of whole-genome sequencing. Phylogenetic analyses of the rotavirus genome reveal repeated intersections between the evolution of human and animal rotavirus strains which is probably a consequence of multiple events of transmission among various animal species. The interspecies transmission and subsequent gene reassortment are important mechanisms driving the diversity of rotaviruses and enabling the emergence of new pathogenic strains.

Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains.

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.