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.

Genotyping of noroviruses from patients of the Pilsen University Hospital in the Czech Republic, 2017-2020.

OBJECTIVE: Noroviruses are members of the Caliciviridae family and are currently segregated into at least 10 genogroups. The distribution of these viruses in the Czech Republic has not yet been investigated in detail. A pilot study was performed to contribute to the overall knowledge and understanding of norovirus circulation in the population. Clinical specimens from patients diagnosed with norovirus infection during their hospitalization at the Pilsen University Hospital were genotyped. MATERIAL AND METHODS: A total of 118 patients were diagnosed with norovirus infection between July 2017 and March 2020. Stool samples from patients presenting with gastroenteritis were routinely screened by the RIDA®QUICK Norovirus Test (R-Biopharm AG), a rapid chromatographic immunoassay for the qualitative detection of Norovirus genogroups I and II, according to the manufacturers instructions. Norovirus positive samples were subsequently analysed by molecular biological methods. Stool suspensions (10%) were prepared with phosphate-buffered saline, and nucleic acid was extracted using the QIAamp Viral RNA kit (Qiagen) according to the manufacturers instructions. To investigate the genotype distribution, RT-PCR targeting specific sections of the norovirus genome (region C) was employed, followed by purification of PCR products using the QIAquick PCR Purification Kit (Qiagen) and sequencing (Eurofins Genomics). The sequences obtained were analysed by the MEGA X software, and the results of phylogenetic analyses were confirmed by Norovirus Typing Tool Version 2.0 (https://www.rivm.nl/mpf/typingtool/ norovirus/. RESULTS: During the study period, 14 norovirus genotypes or genogroup variants NoV GI, NoV GII and NoV GIX (previously NoV GII.15) were identified. The major genotype NoV GII.4 Sydney-2012 accounted for a total of 70.3% of norovirus gastroenteritis cases. This norovirus variant was detected in 24 months out of 34 months of the study period. In general, the number of norovirus infections increased during autumn and winter months (October to March) when 68 (57.6%) cases were diagnosed. Although norovirus infection was confirmed in all age categories (age range 0-96 years, median 8, mean 27.9), the statistical analysis revealed a significant difference in the incidence of NoV GII.4 infection between the age group 0-4 years and older patients (χ2 = 3.95, P = 0.047). Many patients (51) were residents of the Pilsen-city district. The case history data showed that 35 of them (29.7%) had another family member who developed symptoms of gastroenteritis at the time of the onset of their infection. CONCLUSION: The pilot study is the first attempt to map the molecular epidemiology of noroviruses, not only in the Pilsen Region but also in the whole Czech Republic. Despite the relatively low number of officially reported cases, noroviruses are undoubtedly one of the most important causes of gastroenteritis in this country. Further studies are therefore necessary to expand the body of knowledge of their ecology and circulation.