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African Swine Fever Virus (ASFV), resulting in strain-dependent vascular pathology, leading to hemorrhagic fever, is an important pathogen in swine. The pathogenesis of ASFV is determined by the array and spatial distribution of susceptible cells within the host. In this study, the replication characteristics of ASFV genotype I E70 (G1-E70) and ASFV genotype II Belgium 2018/1 (G2-B18) in the environment of small veins were investigated in an established vein explant model. Immunofluorescence staining analysis revealed that perivenous macrophages (CD163 + cells) were widely distributed in the explant, with most of them (approximately 2-10 cells/0.03 mm 2 ) being present close to the vein (within a radius of 0-348 µm). Upon inoculation with G1-E70 and G2-B18, we observed an increase in the quantity of cells testing positive for viral antigens over time. G1-E70 replicated more efficiently than G2-B18 in the vein explants (7.6-fold for the ear explant at 72 hpi). The majority of ASFV + cells were CD163 + , indicating that macrophages are the primary target cells. Additional identification of cells infected with ASFV revealed the presence of vimentin + , CD14 + , and VWF + cells, demonstrating the cellular diversity and complexity associated with ASFV infection. By the use of this new vein explant model, the susceptibility of vascular and perivascular cells to an ASFV infection was identified. With this model, it will be possible now to conduct more functional analyses to get better insights into the pathogenesis of ASFV-induced hemorrhages.

African swine fever virus (ASFV) is a substantial threat to pig populations worldwide, contributing to economic disruption and food security challenges. Its spread is attributed to the oronasal transmission route, particularly in animals with acute ASF. Our study addresses the understudied role of nasal mucosa in ASFV infection, using a nasal explant model. The explants remained viable and revealed a discernible ASFV infection in nasal septum and turbinates post-inoculation. Interestingly, more infected cells were found in the turbinates despite its thinner structure. Further analyses showed (i) a higher replication of genotype II strain BEL18 than genotype I strain E70 in the epithelial cell layer, (ii) a preference of ASFV infection for the lamina propria and a tropism of ASFV for various susceptible cell types in different areas in the nasal mucosa, including epithelial cells, macrophages, and endothelial cells. Using porcine respiratory epithelial cells (PoRECs), isolated from nasal tissue, we found a difference in infection mechanism between the two genotypes, with genotype I favoring the basolateral surface and genotype II preferring the apical surface. Moreover, disruption of intercellular junctions enhanced infection for genotype I. This study demonstrated that ASFV may use the respiratory mucosa for entry using different cell types for replication with a genotype difference in their infection of respiratory epithelial cells., (© 2023. The Author(s).)

Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary.

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

The TCR delta enhancer (Edelta) and TCR alpha enhancer (Ealpha) play critical roles in the temporal and lineage-specific control of V(D)J recombination and transcription at the TCR alphadelta locus, working as a developmental switch controlling a transition from TCR delta to TCR alpha activity during thymocyte development. Previous experiments using a transgenic reporter substrate revealed that substitution of the 116-bp minimal Ealpha, denoted Talpha1-Talpha2, for the entire 1.4-kb Ealpha led to a premature activation of V(D)J recombination. This suggested that binding sites outside of Talpha1-Talpha2 are critical for the strict developmental regulation of TCR alpha rearrangement. We have further analyzed Ealpha to better understand the mechanisms responsible for appropriate developmental regulation in vivo. We found that a 275-bp Ealpha fragment, denoted Talpha1-Talpha4, contains all binding sites required for proper developmental regulation in vivo. This suggests that developmentally appropriate enhancer activation results from a functional interaction between factors bound to Talpha1-Talpha2 and Talpha3-Talpha4. In support of this, EMSAs reveal the formation of a large enhanceosome complex that reflects the cooperative assembly of proteins bound to both Talpha1-Talpha2 and Talpha3-Talpha4. Our data suggest that enhanceosome assembly is critical for developmentally appropriate activation of Ealpha in vivo, and that transcription factors, Sp1 and pCREB, may play unique roles in this process.

The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.