The surface modification of the silica-coated magnetic nanoparticles and their application in molecular diagnostics of virus infection.

The study presents a series of examples of magnetic nanoparticle systems designed for the diagnosis of viral diseases. In this interdisciplinary work, we describe one of the most comprehensive synthetic approaches for the preparation and functionalization of smart nanoparticle systems for rapid and effective RT-PCR diagnostics and isolation of viral RNA. Twelve different organic ligands and inorganic porous silica were used for surface functionalization of the Fe3O4 magnetic core to increase the number of active centres for efficient RNA binding from human swab samples. Different nanoparticle systems with common beads were characterized by HRTEM, SEM, FT-IR, XRD, XPS and magnetic measurements. We demonstrate the application of the fundamental models modified to fit the experimental zero-field cooling magnetization data. We discuss the influence of the nanoparticle shell parameters (morphology, thickness, ligands) on the overall magnetic performance of the systems. The prepared nanoparticles were tested for the isolation of viral RNA from tissue samples infected with hepatitis E virus-HEV and from biofluid samples of SARS-CoV-2 positive patients. The efficiency of RNA isolation was quantified by RT-qPCR method.

Genetic variability of pig and human rotavirus group A isolates from Slovakia.

The aim of this work was the genetic typing of RVA isolates originating from pigs and human patients in Slovakia. Seventy-eight rectal swabs from domestic pigs and 30 stool samples from humans were collected. The whole VP7 (G genotypes), VP6 (I genotypes) and partial VP4 (P genotypes) ORFs were amplified by RT-PCR. Genetic variability was higher amongst porcine sequences, where four G genotypes (G3, G4, G5, G11), two P genotypes (P[6], P[13]) and one I5 genotype were detected. Human RVA strains were represented by two G genotypes (G1, G3), two I genotypes (I1, I2), and one P genotype (P[8]). Genetic analysis did not show a relationship between Slovakian porcine and human RVA strains, but phylogenetic grouping of some Slovakian porcine sequences with Hungarian human sequences in both G and P genotypes was observed.

Genetic variability of the S1 subunit of enteric and respiratory bovine coronavirus isolates.

Bovine coronavirus (BCoV) is considered an important pathogen in cattle worldwide. It is a causative agent of enteric and respiratory diseases of cattle. The S1 subunit of the viral S glycoprotein is responsible for virus binding to host-cell receptors, induction of neutralizing antibody and hemagglutinin activity. The entire S1 genomic region (2304 bp) of two enteric bovine coronavirus isolates from Austria, one respiratory and one enteric isolate from Slovakia were analyzed at the genetic level. The comparative analysis of those four isolates revealed 97.1-98.6% similarity at the nucleotide and 95.6-98.6% at the amino acid level. No differences between enteric and respiratory isolates were observed at the genetic level. The isolates were clustered in the phylogenetic tree with European isolates independently of their enteric or respiratory origin.

Genetic typing of porcine reproductive and respiratory syndrome virus isolates from central European countries.

All thirty-three but one porcine reproductive and respiratory syndrome virus (PRRSV) isolate originating from pigs in Austria, Czech Republic and Slovakia were typed on the basis of partial ORF5 sequence as PRRSV-1, subtype EU-1. The single isolate of PRRSV-2 originated from Slovakia.

Genetic characterization of a border disease virus isolate originating from Slovakia.

In this study, a major part of genome of the pestivirus isolate 297 from Slovakia, comprising the 7195 nt-long 5΄-UTR-NS3 region was sequenced and analyzed. Conserved cleavage sites between individual viral proteins of this region were determined and the number of amino acids of respective proteins was estimated as follows: 168 for Npro, 100 for C, 227 for Erns, 195 for E1, 373 for E2, 70 for p7, 453 for NS2, and 683 for NS3. Based on sequence and phylogenetic analysis of 5΄-UTR, Npro, and E2 the isolate 297 was characterized as a border disease virus of genotype 3. It was found to be distinct from other BDV-3 strains analyzed so far, consequently forming a distinct branch within the phylogenetic clade. All these data expand a relatively limited knowledge of genetic properties of individual BDV genotypes and strains circulating in the Central Europe.

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia.

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n=10) into two known genotypes, PCV-2a (n=1) and PCV-2b (n=9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.

Genetic analysis of a bovine viral diarrhea virus 2 isolate from Slovakia.

The identification and genetic characterization of bovine viral diarrhea virus (BVDV) isolate 17237 detected in western Slovakia is described. The analysis of 5'-untranslated region (5'-UTR), autoprotease (Npro) gene, and structural genes (C, Erns, E1, E2) was carried out. The percentage of nucleotide and deduced amino acid identity in analyzed genes implied that the isolate was closely related to the bovine viral diarrhea virus 2 (BVDV-2). Furthermore, the phylogenetic analysis revealed that this isolate fall into BVDV-2b subtype that is sporadic in Europe. The cleavage sites between viral proteins were similar to the ones of a reference strain of BVDV-2.

Cumulation NOx in foodstuffs and raw materials of animal origin.

In recent years quality of all raw materials processed by agricultural-food industry complex has been influenced by penetration of contaminating hazardous substances into the food chain as a result of high chemization, as well as by the exhalation fall-outs of industrial enterprises. In our study we followed the cumulation of nitrates and nitrites in food and raw materials of animal origin in the exposed industrial area of East Slovak Ironworks in Kosice. Determined were the residual levels of nitrates and nitrites in meat and organs of slaughterhouse cattle, milk and water from individual agricultural enterprises. Consistent control of the content of these hazardous substances in foodstuffs of vegetable and animal origin, water, soil and air represents a considerable contribution from viewpoint of consumer protection.

[Dynamics of methemoglobin levels in the blood of calves in relation to the ingested quantity of nitrates and the transrenal transport of nitrates]. / Dynamika hladín methemoglobínu v krvi teliat v závislosti na mnozstve prijatých dusicnanov a transrenálny prestup dusicnanov.

Changes of methaemoglobin levels were investigated in the blood of suckling calves. Transrenal passage of nitrates was determined in dependence on the ingested amount of nitrates. The experiments were conducted under defined husbandry conditions; no excessive nitrate and nitrite supplementation of the calves by feeds and water could be stated. Imitation of possible field conditions when mainly water, but feeds too, may contain higher nitrate and nitrite levels, was carried out by peroral administration of an aquaeous solution of KNO3 to calves. The administered dose was increased from one to 2, 5 and 10 g per animal and day, respectively, in weekly intervals. MtHb determination in the blood of experimental calves on day 1 and 5 of the administration of 1 g KNO3 revealed no significant values. On day 1 and 5 of the administration of 2, 5 and 10 g KNO3 per animal and day, respectively, a significant increase of MtHb levels in the blood of calves was observed 2 and 3 hours after administration, followed by a decrease 4 hours after administration. The maximum values of MtHb in the blood of experimental calves, observed 3 hours following application of the respective KNO3 dose, were within the tolerance limits of the reference values. In urine, 3 hours after the administration of 10 g of KNO3 a mean nitrate value of 941.40.