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.

Prevalence of Mycobacterium avium subspecies paratuberculosis and hepatitis E in New World camelids in Austria.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis in domestic ruminants and New World Camelids (NWC). Hepatitis E virus (HEV) is an important public health concern worldwide. The virus has been identified in several species, some of them serving as a reservoir for zoonotic HEV strains. Husbandry and breeding of llamas and alpacas have increased in Austria in recent years. Therefore, the aim of the present study was to evaluate the prevalence of MAP and HEV in NWC in Austria. Altogether 445 animals, originating from 78 farms were enrolled in the study. Of the animals sampled, 184 (41.35%) were llamas and 261 (58.65%) were alpacas. 443 blood samples for MAP-ELISA and 399 faecal samples for quantitative PCR (qPCR) and culture for MAP as well as for HEV detection by RT-qPCR have been collected. All of the 399 animals tested for shedding of MAP were negative by faecal solid culture. Using qPCR, 15 (3.8%) of the animals were MAP positive and 384 (96.2%) negative. Out of the 443 serum samples examined for specific antibodies against MAP by ELISA, 6 (1.4%) were positive, 1 (0.2%) was questionable and 436 (98.4%) samples were negative. All faecal samples were tested negative for HEV.

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains.

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1 %, 18/64), followed by that of porcine (15.4 %, 6/39) and bovine (5.1 %, 2/39) origins. Hepatitis E virus (5 %, 1/20) and noroviruses GII (14.3 %, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3 %, 9/108), followed by that of porcine, 4.5 % (4/89) and bovine, 1.1 % (1/89) origins. Norovirus GII (3.6 %, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.

[Hepatitidis E virus]. / Virus hepatitidy E.

UNLABELLED: The number of detected cases of hepatitis E (HE) shows an increasing trend in the Czech Republic, probably due the introduction of new diagnostic methods and greater awareness of HE. In most cases, the source of infection has remained unconfirmed or entirely unclear. The causative agent, the hepatitis E virus (HEV), belongs to the Hepeviridae family. Recently, many isolates from mammals, birds, and fish have been identified. HEV is widespread among farm pigs and wild boar populations all over the word. Both species serve as HEV reservoirs. Consumption of undercooked meat or offal from the reservoir animals is considered to be unsafe due to zoonotic transmission of HEV. The aim of the present article is to summarize recent findings regarding HEV. KEY WORDS: hepatitis E - zoonosis - food-borne infection.

[Retrospective NGS Study in High-risk Hereditary Cancer Patients at Masaryk Memorial Cancer Institute]. / Retrospektivní NGS studie u vysoce rizikových pa-cientu s hereditární predispozicí k nádorovému onemocnení v Masarykove onkologickém ústavu.

BACKGROUND: Currently, more than 200 hereditary cancer syndromes have been described, yet, in most countries genetic testing is restricted to a narrow spectrum of genes within a limited group of people tested. METHODS: For this retrospective study we used the TruSight cancer panel (Illumina)--NGS panel targeting 94 cancer predisposition genes in order to analyze 50 high-risk cancer patients with significant personal and family history of cancer who did not carry mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6, TP53 or APC genes. All pathogenic and potentially pathogenic mutations detected by NGS technology have been confirmed by Sanger sequencing. RESULTS: There were several deleterious (frame-shift/nonsense) mutations detected in ATM, BAP1, FANCC, FANCI, PMS2, SBDS, ERCC2, RECQL4 genes. Various pathogenic or potentially pathogenic (missense, predicted splice site, in-frame insertion/deletion) mutations were detected in ATM, BRIP1, CDH1, CHEK2, ERCC2, ERCC3, ERCC4, FANCA, MC1R, MEN1, MRE11A, MUTYH, PALB2, RAD51C, RET, SDHB, STK11. These mutations affect highly conserved protein domains and affect their function as proved by the available functional assays. They were confirmed to be pathogenic as an "Parent No2 " in serious recessive diseases such as Ataxia telangiectasia or Fanconi anemia. The clinical significance of the majority of detected missense variants still remains to be identified. CONCLUSION: Moderate or low penetrance variants are of limited clinical importance. Panel genetic testing in high-risk individuals with cancer provides important information concerning the cause of the investigated cancer, and may assist in the risk assesment and optimal management of the cancer, as well as in further preventive care.

Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens.

RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this review.

[The clinical importance of a genetic analysis of moderate-risk cancer susceptibility genes in breast and other cancer patients from the Czech Republic]. / Klinický význam analýz genu stredního rizika pro hodnocení rizika vzniku karcinomu prsu a dalsích nádoru v Ceské republice.

BACKGROUND: Analysis of the major breast cancer (BC) predisposition genes BRCA1 and BRCA2 enables identification of high-risk individuals. Specialized programs enrolling the carriers of BRCA1/2 mutations facilitate an improvement in prevention and early diagnostics in asymptomatic individuals and rationalize the selection of individualized treatment in case of a BC onset. However, the carriers of mutations in the major predisposition genes represent only approximately 25% of cases among high-risk BC patients. Numerous candidate predisposing genes for breast and other cancers have recently been identified. The risk of cancer development associated with alterations in these genes is lower, and there is a considerable population variability in different regions worldwide. AIM: We have performed mutation analyses of moderate-risk cancer susceptibility genes to evaluate their clinical importance for genetic counseling in high-risk patients suffering from breast and other cancers in the Czech population. RESULTS: Czech oncological patients were analysed for mutation in ATM, CHEK2, NBS1 (NBN) and PALB2 genes. The majority of analyzed individuals represent the population of high-risk BRCA1/2-negative BC patients. CONCLUSIONS: Based on results of this study, we recommend an analysis of recurrent truncating mutations in the CHEK2 gene (the c.1100delC mutation and a large deletion affecting exons 9-10) in BRCA1/2-negative patients from high-risk BC families. A clinical assessment of missense variants in CHEK2 is not suitable. A routine mutation analysis of the ATM and NBS1 (NBN) genes is not recommended in BC patients due to the low frequency of alterations in these genes in the Czech Republic. An identification of truncating mutations in the PALB2 gene is important in BRCA1/2-negative BC patients from families with a strong history of BC (HBC families). The frequency of PALB2 mutations may be comparable to the frequency of mutations in the BRCA2 gene in Czech HBC families.

Genetic testing and prevention of hereditary cancer at the MMCI--over 10 years of experience.

Hereditary cancer syndromes are frequently seen in young cancer patients and patients with a positive family history. Genetic testing is important for the identification of high-risk individuals, and for the early introduction of specialized preventive care or prophylactic surgeries. High-risk tumour suppressor genes (BRCA1 and BRCA2) and DNA repair genes (MLH1, MSH2 and MSH6) are responsible for a substantial part of hereditary breast, ovarian and colorectal cancer. Other hereditary cancers are seen less frequently, but genetic testing has increased for many other site-specific cancers and complex syndromes. Genetic centres and molecular genetic laboratories are located mostly within university or regional hospitals. Some genetic centres are private. It is highly recommended (Czech Society for Medical Genetics) that all laboratories are accredited according to ISO 15,189 and that genetic testing of hereditary cancer syndromes is indicated by medical geneticists. The indication criteria and prevention strategies were published in Supplement 22 of Clinical Oncology 2009 (in Czech). Preventive care for high-risk individuals is organized by thirteen Oncology Centres, which provide most of the oncology care in the Czech Republic. Genetic testing and preventive care for high-risk individuals and mutation carriers is covered by health insurance. The molecular genetic laboratory at the MMCI provides molecular genetic testing of BRCA1, BRCA2, CHEK2 for hereditary breast/ovarian cancer, MLH1, MSH2, MSH6 for Lynch syndrome,TP53 for Li-Fraumeni syndrome, CDKN2A for familial malignant melanoma syndrome and CDH1 gene for hereditary diffuse gastric cancer. Other syndromes are tested in specialized laboratories elsewhere.The use of genetic testing is increasing because of more frequent referrals from oncologists and other specialists and the increasing variety of genes tested. However, in some patients the testing is not recommended and other family members are dying because of the late diagnosis of hereditary syndrome. Greater awareness of the importance of genetic testing in oncology is needed.

Detection and genetic characterisation of Hepatitis E virus in Czech pig production herds.

The objective of the present study was to carry out a small surveillance programme in Czech pig production herds using the nested reverse transcriptase-polymerase chain reaction (nRT-PCR) technique to trace Hepatitis E virus (HEV) in different biological samples and to characterise the detected swine HEV isolates by phylogenetic analysis. A total of 32 piglets from 11 herds clinically suspected of postweaning multisystemic wasting syndrome (PMWS) were examined. Bile, liver tissue and serum samples were collected from each animal. Due to the high genetic variability of HEV, three sets of primers targeting each of the open reading frames (ORFs) of its genome were used. HEV RNA was most frequently detected in the bile samples (40.0%), followed by liver tissue (16.1%) and serum (3.2%). Seven (63.6%) of the 11 monitored farms were found to have at least one HEV RNA positive piglet. Specific 242 bp sequences within the ORF1 coding non-structural proteins were sequenced and phylogenetically analysed. Phylogenetic analysis using the neighbor-joining and maximum parsimony method confirmed that all detected Czech swine HEV isolates belonged to genotype III. Comparison of the Czech swine HEV isolates with corresponding sequences of swHEV available in GenBank failed to find any 100% homologous HEV isolate.