MicroRNAs participate in the regulation of apoptosis and oxidative stress-related gene expression in rabbits infected with Lagovirus europaeus GI.1 and GI.2 genotypes.

MicroRNAs (miRs) are a group of small, 17-25 nucleotide, non-coding RNA that regulate gene expression at the post-transcriptional level. To date, little is known about the molecular signatures of regulatory interactions between miRs and apoptosis and oxidative stress in viral diseases. Lagovirus europaeus is a virus that causes severe disease in rabbits (Oryctolagus cuniculus) called Rabbit Hemorrhagic Disease (RHD) and belongs to the Caliciviridae family, Lagovirus genus. Within Lagovirus europaeus associated with RHD, two genotypes (GI.1 and GI.2) have been distinguished, and the GI.1 genotype includes four variants (GI.1a, GI.1b, GI.1c, and GI.1d). The study aimed to assess the expression of miRs and their target genes involved in apoptosis and oxidative stress, as well as their potential impact on the pathways during Lagovirus europaeus-two genotypes (GI.1 and GI.2) infection of different virulences in four tissues (liver, lung, kidneys, and spleen). The expression of miRs and target genes related to apoptosis and oxidative stress was determined using quantitative real-time PCR (qPCR). In this study, we evaluated the expression of miR-21 (PTEN, PDCD4), miR-16b (Bcl-2, CXCL10), miR-34a (p53, SIRT1), and miRs-related to oxidative stress-miR-122 (Bach1) and miR-132 (Nfr-2). We also examined the biomarkers of both processes (Bax, Bax/Bcl-2 ratio, Caspase-3, PARP) and HO-I as biomarkers of oxidative stress. Our report is the first to present the regulatory effects of miRs on apoptosis and oxidative stress genes in rabbit infection with Lagovirus europaeus-two genotypes (GI.1 and GI.2) in four tissues (liver, lungs, kidneys, and spleen). The regulatory effect of miRs indicates that, on the one hand, miRs can intensify apoptosis (miR-16b, miR-34a) in the examined organs in response to a viral stimulus and, on the other hand, inhibit (miR-21), which in both cases may be a determinant of the pathogenesis of RHD and tissue damage. Biomarkers of the Bax and Bax/Bcl-2 ratio promote more intense apoptosis after infection with the Lagovirus europaeus GI.2 genotype. Our findings demonstrate that miR-122 and miR-132 regulate oxidative stress in the pathogenesis of RHD, which is associated with tissue damage. The HO-1 biomarker in the course of rabbit hemorrhagic disease indicates oxidative tissue damage. Our findings show that miR-21, miR-16b, and miR-34a regulate three apoptosis pathways. Meanwhile, miR-122 and miR-132 are involved in two oxidative stress pathways.

A Feedback Control Sensing System of an Electrorheological Brake to Exert a Constant Pressing Force on an Object.

The paper presents the application of a strain gauge sensor and a viscous brake filled with an electrorheological (ER) fluid, which is a smart material with controlled rheological properties, by an electric field to the fluid domain. For experimental tests, a cylindrical viscous brake was designed. The tests were carried out on a test stand especially prepared for this purpose and suitable for the examination of the impact of the rotational speed of the input shaft and the value of the electric voltage supplied to the viscous brake on pressing forces, taking into account the ER fluid temperature and brake fluid filling level. On the basis of the experimental research results, a viscous brake control system to exert constant pressing forces with feedback from a strain gauge sensor, based on the programmable logic controller, was designed and implemented. This system, using its own control algorithm, ensured a control pressing force within the assumed range, both during the constant and follow-up control. The measurement results obtained during the tests of the viscous brake designed to exert a force were presented in the form of time courses, showing the changes of the pressing force, the electric voltage applied to the brake and the rotational speed of the brake input shaft. The developed ER fluid brake control system with feedback was tested for constant and follow-up control, taking into account the impact of the working fluid temperature. During the test it was possible to obtain a maximum pressing force equal to 50 N for an electric voltage limited to 2.5 kV. The resultant error was lower than 1 N, wherein the adjustment time after changing the desired value of the force was around 1.5 s. The correct operation of both the brake and the control system, as well as the compatibility of the pressing force value and time adjustment, were determined. The main technical contribution described in this article is the design of a new type of DECPF and a new method for its control with the use of a specifically programmed programmable logic controller which simulates the proportional-integral controllers' operation.

European Brown Hare Syndrome in Poland: Current Epidemiological Situation.

European brown hare syndrome (EBHS) is one of the main causes of mortality in brown hares (Lepus europaeus) and mountain hares (Lepus timidus) in Europe. Since the mid-1990s, this highly lethal and contagious plague has been widespread in many European countries, contributing to a drastic decline in the number of free-living and farmed hares. A second lagovirus, able to infect some species of hares is rabbit haemorrhagic disease virus 2 (RHDV2; GI.2) recognised in 2010, a new viral emergence of RHDV (GI.1) which is known to be responsible for haemorrhagic disease in rabbits-RHD. The aim of this study was to evaluate the current EBHS epidemiological situation on the basis of the presence of antibodies to European brown hare syndrome virus (EBHSV) and anti-RHDV2 antibodies in sera collected from free-ranging hares in Central and Southeastern Poland in 2020-2021. Additionally, studies on the presence of EBHSV and RHDV2 antigens or their genetic material in the blood and internal organs taken from brown hares between 2014 - 2021 have been carried out. The results of the serological examination showed nearly 88% of tested blood samples were positive for EBHSV antibodies. No EBHSV was identified in the examined hares using virological and molecular tests. The positive results of EBHS serological studies confirmed the circulation and maintenance of EBHSV in free-living brown hares in Poland. However, no serological, virological or molecular evidence was obtained indicating that the brown hares tested had been in contact with RHDV2.

Phylogenetic Analysis of European Brown Hare Syndrome Virus Strains from Poland (1992-2004).

European brown hare syndrome (EBHS) is lethal to several species of free-living hares worldwide. The genetic characterization of its virus (EBHSV) strains in European circulation and epidemiological knowledge of EBHSV infections is not yet complete. The study determined the nucleotide sequences of the genomes of EBHSV strains from Poland and analyzed their genetic and phylogenetic relationships to a group of hare lagoviruses. The genome of five virus strains detected in Poland between 1992 and 2004 was obtained by RT-PCR and sequencing of the obtained amplicons. The genetic relationships of the EBHSV strains were analyzed using the full genome and VP60 gene sequences. Additionally, the amino acid sequence of the VP60 gene was analyzed to identify mutations specific to recognized EBHSV subgroups. Partial amplification of the virus open reading frame (ORF)1 and ORF2 regions obtained nearly complete nucleotide genome sequences of the EBHSV strains. Phylogenetic analysis placed them in a GII.1 cluster with other European strains related to nonpathogenic hare caliciviruses. VP60 gene analysis allocated these EBHSV strains to the G1.2, G2.2-2.3 or G3 virus genetic groups. The amino acid sequence differences in the entire genome ranged from 1.1 to 2.6%. Compared to a reference French EBHSV-GD strain, 22 variable amino acid sites were identified in the VP60 region of the Polish strains, but only six were in VP10. Single amino acid changes appeared in different sequence positions among Polish and other European virus strains from different genetic groups, as well as in VP10 sequences of nonpathogenic hare caliciviruses. The results of the study showed a high genetic homogeneity of EBHSV strains from Poland despite their different location occurrence and initial detection times. These strains are also phylogenetically closely related to other EBHSV strains circulating in Europe, likely confirming the slow evolutionary dynamics of this lagovirus species.

Evidence of independent introductions of RHDV2 strains in Poland based on the genome analysis of viral isolates from 2016-2018.

The aim of this study was the molecular epidemiology of independently introduced RHDV2 strains in Poland. The nucleotide sequences of RHDV2 diagnosed in domestic rabbits in 2018 in the voivodeships of Swietokrzyskie (strain PIN), Malopolskie (strain LIB) and Mazowieckie (strain WAK), and RHDVa from 2015 (strain F77-3) recognized in wild rabbits in Kujawsko-Pomorskie voivodeship were compared to the genome sequences of the first native RHDV2 strains from 2016-2017. The reference sequences available in public databases, the representative for a classical RHDV (G1-G5 genogroups), RHDVa (G6), non-pathogenic caliciviruses (RCV, GI.3 and GI.4) as well as original and recombinant RHDV2 isolates were included for this analysis. Nucleotide sequence similarity among the most distanced RHDV2 strains isolated in Poland in 2018 was from 92.3% to 98.2% in the genome sequence encoding ORF1, ORF2 and 3'UTR, between 94.8-98.7% in the VP60 gene and between 91.3-98.1% in non-structural proteins (NSP) region. The diversity between three RHDV2 and RHDVa from 2015 was up to 16.3% in the VP60 region. Similarities are shown for the VP60 tree within the RHDV2 group, however, the nucleotide analysis of NSP region revealed the differences between older and new native RHDV2 strains. The Polish RHDV2 isolates from 2016-2017 clustered together with RHDV G1/RHDV2 recombinants, first identified in the Iberian Peninsula in 2012, while all strains from 2018 are close to the original RHDV2. The F77-3 strain clustered to well supported RHDVa (G6) genetic group, together with other Polish and European RHDVa isolates. Based on the results of phylogenetic characterization of RHDV2 strains detected in Poland between 2016-2018 and the chronology of their emergence it can be concluded that RHDV2 strains of 2018 and RHDV2 strains of 2016-2017 were introduced independently thus confirming their different origin and simultaneous pathway of spreading.

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.