The Production of Recombinant African Swine Fever Virus Lv17/WB/Rie1 Strains and Their In Vitro and In Vivo Characterizations.

Lv17/WB/Rie1-Δ24 was produced via illegitimate recombination mediated by low-dilution serial passage in the Cos7 cell line and isolated on PAM cell culture. The virus contains a huge ~26.4 Kb deletion in the left end of its genome. Lv17/WB/Rie1-ΔCD-ΔGL was generated via homologous recombination, crossing two ASFV strains (Lv17/WB/Rie1-ΔCD and Lv17/WB/Rie1-ΔGL containing eGFP and mCherry markers) during PAM co-infection. The presence of unique parental markers in the Lv17/WB/Rie1-ΔCD-ΔGL genome indicates at least two recombination events during the crossing, suggesting that homologous recombination is a relatively frequent event in the ASFV genome during replication in PAM. Pigs infected with Lv17/WB/Rie1-Δ24 and Lv17/WB/Rie1/ΔCD-ΔGL strains have shown mild clinical signs despite that ASFV could not be detected in their sera until a challenge infection with the Armenia/07 ASFV strain. The two viruses were not able to induce protective immunity in pigs against a virulent Armenia/07 challenge.

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune-pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig's immune system. A better understanding of these immune-pathological mechanisms would help to design suitable vaccines against this disease.

Involvement of the MGF 110-11L Gene in the African Swine Fever Replication and Virulence.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease that causes extensive economic and animal welfare losses in the Eurasian pig (Sus scrofa) population. To date, no effective and safe vaccines have been marketed against ASF. A starting point for vaccine development is using naturally occurring attenuated strains as a vaccine base. Here, we aimed to remove the multigene family (MGF) 110 gene of unknown function from the Lv17/WB/Rie1 genome to improve the usability of the virus as a live-attenuated vaccine, reducing unwanted side effects. The MGF 110-11L gene was deleted using the CRISPR/Cas9 method, and the safety and efficacy of the virus were tested in pigs after isolation. The vaccine candidates administered at high doses showed reduced pathogenicity compared to the parental strain and induced immunity in vaccinated animals, although several mild clinical signs were observed. Although Lv17/WB/Rie1/d110-11L cannot be used as a vaccine in its current form, it was encouraging that the undesirable side effects of Lv17/WB/Rie1 at high doses can be reduced by additional mutations without a significant reduction in its protective capacity.

Application of Magnetic Adaptive Testing for Nondestructive Investigation of 2507 Duplex Stainless Steel.

Duplex stainless steels are two-phase alloys, which contain ferritic and austenitic phases in their microstructure. Their duplex structure provides exceptional resistance to pitting and chloride stress corrosion cracking, and their strength is about twice that of austenitic stainless steels. Due to their good properties, they are widely used in chemical and petrochemical industries as a base material in pressure vessels, pipelines and containers. Duplex stainless steel samples were nondestructively investigated by measuring sets of magnetic minor hysteresis loops using the method called magnetic adaptive testing (MAT). Several series of heat-treated and cold-rolled 2507 duplex stainless steels were measured, and the magnetic parameters were compared with the results of the DC magnetometry of the samples. It was found that the changes in the material properties that were generated by heat treatment and mechanical deformation could easily be followed by magnetic measurements. In contrast to DC magnetic measurements, good correlation was found with the magnetic parameters determined by MAT method and Vickers hardness. Based on our experiments, MAT seems to be a powerful tool for the nondestructive characterization of duplex stainless steels.

Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV.

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method.

Combined Short and Long-Read Sequencing Reveals a Complex Transcriptomic Architecture of African Swine Fever Virus.

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS's limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps was also discovered.

Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody.

Following the introduction of African swine fever virus (ASFV) into Europe in 2007, ASFV infection has spread continuously over the past years and it became a high level disease threat in Europe and also Asia. Examination of suspect clinical cases for ASF with rapid and sensitive laboratory methods can substantially contribute to the detection and characterization of new outbreaks. In this study two sensitive tests were developed for the detection of the p72 major capsid protein of ASFV both in cell culture with an immunocytochemical (IC) and in tissue samples with an immunohistochemical (IHC) method using a commercially available mouse monoclonal antibody (clone 1BC11). The IC test was able to detect the virus at high virus dilutions in cell culture and the IHC test indicated the presence of ASFV in all formalin-fixed and paraffin-embedded tissue samples collected from two wild boars. The reported IC and IHC methods were found to be useful ancillary laboratory tests for research purposes and for the diagnosis of acute ASF.

Vaccine Protection Against Experimental Challenge Infection with a PPV-27a Genotype Virus in Pregnant Gilts.

BACKGROUND/INTRODUCTION: Porcine parvovirus (PPV), the causative agent of severe reproductive failures in pigs, is present worldwide. The witnessed spread of the virulent 27a type PPV strains since its recognition raised concerns about the efficacy of the available commercial vaccines. METHODS: To address this question, vaccinated pregnant gilts were challenged with a PPV-27a-like virus strain and parameters related to vaccine efficacy were compared. RESULTS: The K22 vaccine strain of Parvoruvax® (PVX) was characterized as "Kresse-like" based on the epitope mapping data. Vaccination of the gilts induced a low level of antibody responses. Based on foetal mortality, the number of sows which had challenge virus-affected foetuses, the percent of PPV positive piglets/litters plus their PPV genome and viral load PVX outscored the other vaccinated groups. CONCLUSION: Stronger protection was provided by the "Kresse-like" K22 PPV strain-based vaccine than by the NADL-2 and NADL-like strain-based commercial vaccines against a PPV-27a cluster strain challenge. Vaccine-induced antibody levels as measured pre-challenge were not found to be an accurate indicator of protection.

A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus.

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

Complex Study of Eutectoidal Phase Transformation of 2507-Type Super-Duplex Stainless Steel.

The aim of this work was to study expansively the process of the eutectoidal phase transformation of 2507-type super-duplex stainless steel. Three sample sets were prepared. The first sample set was made to investigate the effect of the previous cold rolling and heat treatment for the eutectoidal phase transformation. Samples were cold rolled at seven different rolling reductions which was followed by heat treatment at five different temperatures. The second sample set was prepared to determine the activation energy of the eutectoidal decomposition process using the Arrhenius equation. Samples were cold rolled at seven different rolling reductions and were heat treated at the same temperature during eight different terms. A third sample set was made to study how another plastic-forming technology, beside the cold rolling, can influence the eutectoidal decomposition. Samples were elongated by single axis tensile stress and were heat treated at the same temperature. The results of the first and the third sample sets were compared. The rest δ-ferrite contents were calculated using the results of AC and DC magnetometer measurements. DC magnetometer was used as a feritscope device in this work. Light microscope and electron back scattering diffraction (EBSD) images demonstrated the process of the eutectoidal decomposition. The thermoelectric power and the hardness of the samples were measured. The results of the thermoelectric power measurement were compared with the results of the δ-ferrite content measurement. The accurate value of the coercive field was determined by a Foerster-type DC coercimeter device.

Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0⁻1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host's chromosomes.

Cellular localisation of the proteins of region 3 of feline enteric coronavirus.

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflect in vivo stability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

Biology of Porcine Parvovirus (Ungulate parvovirus 1).

Porcine parvovirus (PPV) is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the "new" highly virulent isolates cannot be neutralized effectively by antisera raised against "old" PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology.

The SAT Protein of Porcine Parvovirus Accelerates Viral Spreading through Induction of Irreversible Endoplasmic Reticulum Stress.

The SAT protein (SATp) of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER), and SAT deletion induces the slow-spreading phenotype. The in vitro comparison of the wild-type Kresse strain and its SAT knockout (SAT-) mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT- virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences in the quantity and the localization of CHOP were detected, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of the induction of irreversible ER stress in porcine testis (PT) cell necrosis and viral egress was confirmed by treatment of infected cells by ER stress-inducing chemicals (MG132, dithiothreitol, and thapsigargin), which accelerated the egress and spreading of both the wild-type and the SAT- viruses. UV stress induction had no beneficial effect on PPV infection, underscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a coexpression experiment.IMPORTANCE SATp is encoded by an alternative open reading frame of the PPV genome. Earlier we showed that SATp of the attenuated PPV NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that slow spreading is a general feature of SAT- PPVs and is the consequence of prolonged cell integrity. PPV infection induced ER stress in infected cells regardless of the presence of SATp, as demonstrated by the morphological changes of the ER and expression of the stress response proteins Xbp1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated cell death during infection, as shown by the higher rate of expression of CHOP and alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by treatment of infected cells with ER stress-inducing chemicals.

Detection of Porcine Parvovirus 2 (Ungulate Tetraparvovirus 3) Specific Antibodies and Examination of the Serological Profile of an Infected Swine Herd.

Porcine parvovirus 2 (PPV2) is a member of a recently discovered group of swine parvoviruses occurring worldwide. It is frequently detected in lung samples suggesting some pathological role of the virus in diseases. To study this possibility an indirect ELISA was developed to detect PPV2 specific antibodies and to examine the serological profile of an infected swine herd where 185 serum samples collected from different age groups including sows were analyzed. According to the results maternal antibody levels decreased until 14 days of age and PPV2 specific antibodies started to rise between 28 to 43 days of age when respiratory signs were also observed in the examined swine herd. At 57 days of age the clinical signs disappeared and a rapid increase of PPV2 specific antibody levels could be measured simultaneously, peaking at 57 days of age. The viraemic status of different age groups was determined by qPCR using serum samples. At least a low level of viraemia was measured in every age group, but higher copy number of PPV2 was only detected at 57 days of age and the level decreased in older age groups. The changes in virus load and antibody levels together with the onset and decrease of clinical signs suggested that PPV2 had a role in the development of respiratory signs.

Propagation of viruses infecting waterfowl on continuous cell lines of Muscovy duck (Cairina moschata) origin.

Duck circovirus, duck hepatitis A virus 1, goose parvovirus and goose haemorrhagic polyomavirus are economically damaging pathogens of waterfowl, and replicate poorly or not at all in established cell lines. AGE1.CR, AGE1.CR.pIX and AGE1.CS cell lines, originating from the Muscovy duck, were tested for their suitability to isolate and identify these viruses. Immunofluorescence (IF) and quantitative polymerase chain reaction investigations verified that all cell lines are permissive for all four viruses; however, AGE1.CR.pIX proved to be the most productive and most sensitive for viral infection. IF experiments revealed that the time of one infectious cycle is approximately 12 to 14 h in the AGE1.CR.pIX cells in the case of the three DNA viruses, while it is 10 to 12 h for DHAV-1. Specific viral infectivity and the limit of detection by IF varied between 55 and 1484 copies, depending on the viruses and cell lines. Despite the high sensitivity of the cell lines for viruses, their viral productivity remained relatively low for the investigated field isolates. However, optimization of virus infection and/or the adaptation of the viruses to the cells can raise viral productivity and can make these cell lines suitable for vaccine development and production.

Characterization of a cold-rolled 2101 lean duplex stainless steel.

Duplex stainless steels (DSS) may be defined as a category of steels with a two-phase ferritic-austenitic microstructure, which combines good mechanical and corrosion properties. However, these steels can undergo significant microstructural modification as a consequence of either thermo-mechanical treatments (ferrite decomposition, which causes σ- and χ-phase formation and nitride precipitation) or plastic deformation at room temperature [austenite transformation into strain-induced martensite (SIM)]. These secondary phases noticeably affect the properties of DSS, and therefore are of huge industrial interest. In the present work, SIM formation was investigated in a 2101 lean DSS. The material was subjected to cold rolling at various degrees of deformation (from 10 to 80% thickness reduction) and the microstructure developed after plastic deformation was investigated by electron backscattered diffraction, X-ray diffraction measurements, and hardness and magnetic tests. It was observed that SIM formed as a consequence of deformations higher than ~20% and residual austenite was still observed at 80% of thickness reduction. Furthermore, a direct relationship was found between microstructure and magnetic properties.

CpG distribution and methylation pattern in porcine parvovirus.

Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can't methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.

Para além do capital: rumo a uma teoria da transição / Beyond capital: towards a theory of transition

A presente obra busca trazer uma reflexão crítica sobre o capital em suas formas, engrenagens e mecanismos de funcionamento. Influenciada por Marx, Lukács e Rosa Luxemburgo, a obra de Mészáros pretende ser um desenho crítico e analítico contra o capital e suas formas de controle social. O autor procura ainda empreender uma reflexão sobre a sociabilidade contemporânea e a lógica que a preside...