Consecutive Passages Research Articles

Xenogeneic-free culture of human intestinal stem cells on functional polymer-coated substrates for scalable, clinical-grade stem cell therapy

The need for basement membrane extract (BME) with undefined constituents, such as Matrigel, for intestinal stem cell (ISC) culture in traditional systems poses a significant barrier that must be overcome for the development of clinical-grade, scalable, ready-to-use ISCs. Here, we propose a functional polymer-based xenogeneic-free dish for the culture of intestinal stem cells (XF-DISC), ensuring substantially prolonged maintenance of ISCs derived from 3-dimensional human intestinal organoids (ISCs3D-hIO). XF-DISC enables remarkable expandability, exhibiting a 24-fold increase in cell numbers within 30 days, with long-term maintenance of ISCs3D-hIO for more than 30 consecutive passages (>210 days). In addition, XF-DISC is fully compatible with a cell banking system. Notably, human pluripotent stem cell-derived ISCs3D-hIO cultured on XF-DISC are successfully transplanted into intestinal injury and inflammation mouse models, leading to engraftment and regeneration of damaged mouse intestinal epithelium. As a reliable and scalable xenogeneic-free ISC3D-hIO culture method, XF-DISC is highly promising for the development of regenerative ISC therapy for human intestinal diseases.

First report on genetic characterization of egg drop syndrome 1976 virus in Egypt.

Since its first description in 1991 in Egypt, egg drop syndrome 1976 (EDS-76) virus has received a little attention as a potential cause for the drop in egg production as well as the reduction in egg quality. To date, no studies have been carried out to describe the genetic characteristics of the circulating field EDS-76 virus strains. Thus, the present study was attempted to estimate the emergence of EDS-76 virus in layer flocks and to determine the genetic diversity between the field strains and the vaccine strain 127. During 2022, a total of 5 apparently healthy backyard layer flocks were investigated for the presence of EDS-76 virus infection following complaints of sudden drop in egg production (25-30%), accompanied by high incidence of eggshell defects. EDS-76 virus DNA was detected in the oviduct samples of 4 (80%) flocks by polymerase chain reaction (PCR) assay targeting the hexon gene of the viral capsid. Attempts of viral isolation in duck embryo revealed no embryonic mortality, however, the allantoic fluids of inoculated eggs exhibited a sustained increase in the hemagglutinating (HA) activity throughout three consecutive passages. The obtained strain, designated BH-1, was characterized on the basis of partial hexon gene sequence analysis (GenBank accession number OR531368). The BH-1 strain displayed 99.6% nucleotide identity with the vaccine strain 127. However, amino acid alignments with the vaccine strain 127 revealed that the BH-1 strain carried 5 non-synonymous mutations. In addition, two of these mutations were incorporated into the hexon hypervariable regions (HVRs), which are strictly responsible for eliciting serotype-specific neutralizing antibodies. In conclusion, the present study represents a starting point for genetic characterization of EDS-76 virus in Egypt and highlights the importance for continuous monitoring and characterization of the circulating field EDS-76 virus strains, in order to determine the proper control strategy.

Antimicrobial Resistance Development in vitro: Adaptive Laboratory Evolution Method (Review)

INTRODUCTION. High rates of emergence and spread of antimicrobial resistance (AMR) necessitate the rapid development of novel antibacterial medicinal products. The assessment of the microbial potential for AMR development under controlled conditions in vitro can save resources during drug development and marketing authorisation and contribute to creating the most effective medicinal products.AIM. The aim was to determine the possibility of the use of the adaptive laboratory evolution (ALE) method to study the development of antimicrobial resistance.DISCUSSION. A variety of methods can be used to investigate the mechanisms of AMR and the influence of medicinal products on the evolution of bacteria towards AMR. One of the options is the ALE method. ALE experiments are conducted under controlled conditions with prolonged exposure of microorganisms to an antibacterial agent. ALE experiments can include serial transfers of microorganisms to fresh liquid media or Petri dishes, as well as continuous cultivation of microorganisms in a chemostat. ALE protocols are used to develop resistance to different antibacterial agents and require meticulous control of the experimental conditions. To obtain reliable results in an experiment, it is necessary to identify parameters that may affect AMR development in microorganisms. These parameters include but are not limited to the concentration of the antibacterial agent, the number of consecutive passages, the duration of incubation.CONCLUSIONS. To achieve the necessary conditions for resistant microorganisms to form, it is essential to adhere strictly to ALE setup requirements, such as using antibacterial agents at subinhibitory or dynamically increasing concentrations (relative to the minimum inhibitory concentrations for the ancestral strain), performing a certain number of passages for ≥20 generations, and incubating cultures until the stationary phase. Despite the fact that ALE experiments are rather lengthy, these studies can reduce the potential waste of resources on developing new compounds that may have to be discontinued at the stage of production because of AMR development.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

Establishment and identification of the head kidney cell line of yellowfin seabream (Acanthopagrus latus) and its application in a virus susceptibility study

The yellowfin seabream (Acanthopagrus latus) is a crucial marine resource owing to its economic significance. Acanthopagrus latus aquaculture faces numerous challenges from viral diseases, but a robust in-vitro research model to understand and address these threats is lacking. Therefore, we developed a novel A. latus cell line from head kidney cells called ALHK1. This study details the development, characterisation, and viral susceptibility properties of ALHK cells. This cell line primarily comprises fibroblast-like cells and has robust proliferative capacity when cultured at 28 °C in Leibovitz's L-15 medium supplemented with 10–20% foetal bovine serum. It exhibited remarkable stability after more than 60 consecutive passages and validation through cryopreservation techniques. The specificity of the ALHK cell line's origin from A. latus was confirmed via polymerase chain reaction (PCR) amplification of the cytochrome B gene, and a chromosomal karyotype analysis revealed a diploid count of 48 (2n = 48). Furthermore, the lipofection-mediated transfection efficiency using the pEGFP-N3 plasmid was high, at nearly 40%, suggesting that ALHK cells could be used for studies involving exogenous gene manipulation. In addition, ALHK cells displayed heightened sensitivity to the large mouth bass virus (LMBV), substantiated through observations of cytopathic effects, quantitative real-time PCR, and viral titration assays. Finally, the response of ALHK cells to LMBV infection resulted in differentially expressed antiviral genes associated with innate immunity. In conclusion, the ALHK cell line is a dependable in-vitro platform for elucidating the mechanisms of viral diseases in yellowfin seabream. Moreover, this cell line could be valuable for immunology, vaccine development, and host-pathogen interaction studies.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner.

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 μM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

Design, synthesis, and evaluation of N1,N3-dialkyldioxonaphthoimidazoliums as antibacterial agents against methicillin-resistant Staphylococcus aureus

Increasing antibiotic resistance of bacterial pathogens poses a serious threat to human health worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is among the most deleterious bacterial pathogens owing to its multidrug resistance, necessitating the development of new antibacterial agents against it. We previously identified a novel dioxonaphthoimidazolium agent, c5, with moderate antibacterial activity against MRSA from an anticancer clinical candidate, YM155. In this study, we aimed to design and synthesize several novel cationic amphiphilic N1,N3-dialkyldioxonaphthoimidazolium bromides with enhanced lipophilicity of the two side chains in the imidazolium scaffold and improved antibacterial activities compared to those of c5 against gram-positive bacteria in vitro and in vivo. Our new antibacterial lead, N1,N3-n-octylbenzyldioxonaphthoimidazolium bromide (11), exhibited highly potent antibacterial activities against various gram-positive bacterial strains (MICs: 0.19–0.39 μg/mL), including MRSA, methicillin-sensitive S. aureus, and Bacillus subtilis. Moreover, antibacterial mechanism of 11 against MRSA based on the generation of reactive oxygen species (ROS) was evaluated. Although compound 11 exhibited cytotoxic effects in vitro and lacked a therapeutic index against the HEK293 and HDFa mammalian cell lines, it exhibited low toxicity in the Drosophila animal model. Remarkably, 11 exhibited better in vivo antibacterial efficacy than c5 and the clinically used antibiotic, vancomycin, in SA3-infected Drosophila model. Moreover, the development of bacterial resistance to 11 was not observed after 16 consecutive passages. Therefore, rational design of antibacterial cationic amphiphiles based on ROS-generating pharmacophores with optimized lipophilicity can facilitate the identification of potent antibacterial agents against drug-resistant infections.

Construction of a stable expression HEK293T engineered cell line adapted to high-density suspension culture

The HEK293 cell line has received FDA approval and is increasingly used to produce therapeutic glycoproteins. However, the cell line faces the challenge of long-term expression instability. This problem could be solved by specifically integrating exogenous genes into stable sites and developing a HEK293 cell platform to efficiently and stably express recombinant proteins with large-scale suspension culture. The study uses CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) technologies to integrate a variety of exogenous genes into specific chromosomal sites, such as the hROSA26 site, AAVS1 site, and CCR5 site. We evaluated the stability and expression intensity of exogenous proteins at different insertion sites under suspension culture conditions. A monoclonal cell line expressing enhanced green fluorescent protein (EGFP) is obtained using CRISPR/Cas9-mediated specific integration. The positive monoclonal cell lines express EGFP after 60 consecutive passages, and no significant differences in protein expression titer are observed. These results show that, under suspension culture conditions, the hROSA26 locus could stably express exogenous proteins after 60 generations. HEK293 cell lines integrating HSA (68 kDa) and IFNβ-HSA (89 kDa) genes at the above three sites were obtained respectively using Bxb1-mediated RMCE technology, and batch experiments demonstrated their expression capacity. In summary, a stable and efficient protein expression platform of HEK293 cells is successfully constructed and could be adapted to serum-free suspension culture.

Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens.

Chicken coccidiosis caused by Eimeria spp. can occur on almost all poultry farms, causing huge economic losses to the industry. Genetically manipulated Eimeria parasites as a vaccine vector to deliver viral antigens have been reported. In our preliminary study, transgenic E. acervulina expressing a VP2 gene (Ea-VP2) of the infectious bursal disease virus (IBDV) demonstrated partial protection against IBDV infection. To enhance immune responses, we aimed to increase the VP2 gene copy number in transgenic E. acervulina. In this study, we used a novel plasmid vector carrying a VP2 gene fused with three flag tags and a red fluorescent reporter gene (mCherry). The vector was introduced into Ea-VP2 sporozoites through nucleofection, leading to the generation of Ea-2VP2. Subsequent analysis revealed a notable escalation in the fluorescent rate, increasing from 0.11 to 95.1% following four consecutive passages facilitated by fluorescent-activated cell sorting. Verification via PCR, Western blot, and immunofluorescence confirmed the successful construction of the Ea-2VP2 population. Despite lower fecundity compared to wild-type E. acervulina, Ea-2VP2 maintained immunogenicity. Our research effectively created a transgenic E. acervulina strain transfected sequentially with two copies of the VP2 gene from IBDV. This modification resulted in an increased humoral immune response after primary immunization in chickens. Additionally, it demonstrated a degree of protection within the bursa against IBDV infection. Future studies will focus on further enhancing immune response levels.

Об информационном воздействии юридического голосования на правовые отношения

The article analyzes one of the main functions of legal voting, namely the informational function. The author proposed and examined in detail a staged model of informational impact of legal voting on the legal relations that develop on issues of collective adoption of legally significant decisions. It is concluded that this function is one of the most important functions of the considered legal phenomenon, and its practical implementation involves the consecutive passage of four stages. At the same time, the ideal stage model does not assume optional stages, considering each of them as a mandatory one. In practice, the third stage, namely the perception of collectively made legally significant decision, understanding its meaning, by the persons interested in the results of the voting procedure can fail to occur due to objective and subjective factors.

Вивчення життєздатних бактеріофагів за допомогою активації псевдолізогенного стану Pectobacterium carotovorum

The rare occurrence of viable bacteriophages is an issue in studying bacterial-phage systems of the phytopathogenic bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc). Aim. To obtain viable bacteriophages by activating pseudolysogeny of the Pcc strains. Methods. To obtain bacteriophage F66, isogenic clones of Nalr mutants of strains Pcc 66A and Pcc M2-4 were used. To study the properties of the phage, ultracentrifugation of viral particles, electron microscopy, electrophoretic separation, and comparative restriction analysis of virion DNA were used. Results. It was shown that some isogenic clones of Nalr -mutants of Pcc 66A and Pcc M2-4 can produce viable bacteriophages under conditions of intensive aeration. At the same time, induced phages are able to reproduce on the parent bacterial strains, which they were isolated from (pseudolysogenic response). A pure line was obtained for the phage isolate induced from pseudolysogenic Pcc 66A Nalr by consecutive single colony passages. This phage, named F66, was shown to be temperate and able to lyse and lysogenize strains of pectolytic bacteria isolated from soft rot affected potato tubers. Phage F66 has а virion with the A1 morphotype characterized by an isometric head with an average diameter of 52.0 ± 2.1 nm and a contractile tail with a length of 115.4 ± 3.2 nm. Showing low stability under environmental conditions, phage F66 differs significantly from the temperate phage ZF40 P. carotovorum, which has a similar virion morphology. Compared phages also differ in the restriction fragment patterns obtained using endonucleases HindIII, BamHI, and HpaI. Conclusions. Temperate phage F66 is a novel P. carotovorum virus. The method for activating the pseudolysogenic state proposed in the article is useful for obtaining viruses of phytopathogenic bacteria.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Drug repurposing: insights into the antimicrobial effects of AKBA against MRSA

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4–8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections.

Immunomodulatory-associated gene transcripts to multipotency of bovine amniotic fluid mesenchymal stem cells.

The adnexa fetal tissues are sources of mesenchymal stromal cells (MSCs) due to their noninvasive harvest, with all biological material discarded most of the time. MSCs are a promise regarding to their plasticity, self-renewal, differentiation potentials, immunomodulatory and anti-inflammatory properties, which have made clinical stem cell therapy a reality. The present study aimed to characterize and evaluate the immunomodulation ability of bovine mesenchymal cells collected from bovine amniotic fluid (bAFMSCs) isolated and subjected to sixth consecutive culture passages in vitro. The multilineage properties of the bAFMSCs collections confirmed the ability to undergo adipogenic, chondrogenic and osteogenic differentiation. The mesenchymal gene transcription CD106, CD73, CD29, CD90 and CD166 were detected in bAFMSCs, whereas CD34 and CD45 were not detected. Regarding cytokine mRNA expression, IL2, IL6, INFα, INFβ, INFγ, TNFα and TNFβ were downregulated, while IL10 was highly regulated in all studied passages. The present study demonstrated the immunological properties and multipotency of in vitro bAFMSCs collections, and thus, they can be tested in cattle pathological treatments or multiplication by nuclear transfer cloning.

Effect of shell spacing on mechanical behavior of multi-span soil-steel composite structure

This study analyses the effect of lateral shells on central shell at a different spacing in a multi-span soil-steel composite structure subjected to quasi-static moving loads. The displacements and internal forces of the central shell during consecutive truck passages over the structure are investigated by finite element (FE) analysis. Field measurements from a site in Niemcza, Poland, are used to calibrate input parameters. Next, the simulations for different spacing between the shells are investigated. The backfill soil is modeled as elastic-perfectly plastic, while the shells and sheet piles are linear elastic. The non-linear contact zone between the shell and the soil backfill is assumed to reflect an elastic-plastic constitutive model. The analysis reveals that both vertical and horizontal displacements increase significantly when the ratio of shell spacing to span length is less than 0.5. Maximum stress occurs when the shells are placed adjacent to each other, i.e., without spacing. The stress is almost doubled in this position compared to the reference case—a single-span structure. The shifting of extreme deflections and stress is observed in the direction of truck movement. Nevertheless, the influence of lateral shells on the central shell's performance under moving loads is nearly negligible when the spacing-to-span ratio exceeds 0.5.

Ruxolitinib accelerates influenza A virus adaptation in the Madin-Darby canine kidney (MDCK) cell line.

To investigate the effect of ruxolitinib medium supplement, separately and in combination with trypsin, on influenza A virus (IAV) adaptation and propagation in the Madin-Darby canine kidney (MDCK) cell line. Two consecutive passages of three egg-based IAV strains were performed in the MDCK cell line with medium (a) without additives; (b) with a combination of ruxolitinib and trypsin; (c) with ruxolitinib; and (d) trypsin. Adaptation without a medium additive failed in both passages. After a single passage, the probability of the IAV adaptation was highly significantly influenced by the type of additive (binomial generalized linear model, χ22=23.84, P<0.00001). The highest probability of adaptation was achieved with the combination of ruxolitinib and trypsin, followed by ruxolitinib alone and trypsin. After the two consecutive passages, the influence of the type of medium additive on the probability of virus adaptation was no longer significant. In two of three IAV MDCK-adapted strains, the type of medium additive had no significant influence on virus yields. Ruxolitinib accelerates the adaptation of IAV in the MDCK cell line both individually and together with trypsin.

Metavirome Analysis and Identification of Midge-Borne Viruses from Yunnan Province, China, in 2021.

Midges are widely distributed globally and can transmit various human and animal diseases through blood-sucking. As part of this study, 259,300 midges were collected from four districts in Yunnan province, China, to detect the viral richness and diversity using metavirome analysis techniques. As many as 26 virus families were detected, and the partial sequences of bluetongue virus (BTV), dengue virus (DENV), and Getah virus (GETV) were identified by phylogenetic analysis and PCR amplification. Two BTV gene fragments, 866 bps for the VP2 gene of BTV type 16 and 655 bps for the VP5 gene of BTV type 21, were amplified. The nucleotide sequence identities of the two amplified BTV fragments were 94.46% and 98.81%, respectively, with two classical BTV-16 (GenBank: JN671907) and BTV-21 strains (GenBank: MK250961) isolated in Yunnan province. Furthermore, the BTV-16 DH2021 strain was successfully isolated in C6/36 cells, and the peak value of the copy number reached 3.13 × 107 copies/μL after five consecutive BHK-21 cell passages. Moreover, two 2054 bps fragments including the E gene of DENV genotype Asia II were amplified and shared the highest identity with the DENV strain isolated in New Guinea in 1944. A length of 656 bps GETV gene sequence encoded the partial capsid protein, and it shared the highest identity of 99.68% with the GETV isolated from Shandong province, China, in 2017. Overall, this study emphasizes the importance of implementing prevention and control strategies for viral diseases transmitted by midges in China.

Experimental Epizotology of Low-Virulent Variants of African Swine Fever Virus

African swine fever (ASF) remains an urgent problem of pig farming in Ukraine, the solution of which is possible only on the basis of deep scientific knowledge about the specific driving forces of the epizootic in its specific nozoareal. This is necessary in order to target anti-epizootic measures on the most vulnerable link of the epizootic chain in a specific nozoareal. The aim of the work was to develop a low-budget methodological base for experimental epizootology of low-virulent ASFV variants in Ukraine, in particular, to study the mechanisms of the formation of enzootic areas, quality control of anti-epizootic measures, and evaluation of the effectiveness of the antiviral drugs against them in Ukraine. Methods. Experimental and epizootological studies in the adaptation of suckling piglets to keeping in the biosecurity-level BSL-3 for laboratory animals (Patent UA No. 133248 dated 03/25/2019) were conducted at the laboratory base of the Odesa branch of NSC «IECVM». All procedures with infectious active biological materials in the current order were carried out in the BSL-3 module, built and certified with the assistance of the US Government in UAPRI (Odesa). The ASF agent strain «IECVM/Ternopil/2017» (infectious activity 4.0—7.5lg HAdU50/cm 3) circulating in the Ternopil region in 2017—2020 was used as a test virus. The presence of low-virulence variants of the ASFV pathogen in the studied samples was determined by a bioassay on suckling piglets, followed by three consecutive passages on a stable Vero line of the baby green monkey kidney cells. The isolated ASF virus was identified according to the methods and reagents recommended by the OIE Manual. Results. Intermittent passages «by the founder’s method» of dilutions 10-1 and 10-2 of the ASF virus strain «IECVM/Ternopil/2017» on piglets (n=20) and the culture of porcine alveolar macrophages («ASFVPAM») allowed us to identify highly-, moderately-, and low-virulent variants/clones in its composition. Verifi cation by bioassay on suckling piglets (n=5) of low-virulent clones of the agent, which were stabilized in Vero cell culture («ASFVVero»), showed that after intraperitoneal infection at a dose of 4.25 lgHAdU50/cm 3, they are capable of causing only a non-lethal (within 2 weeks) viral infection with a maximum daily rectal temperature of 39.4±0.22 °C and duration of fever on average 1.6±0.14 days (5 of 5 piglets). Clones with greater virulence («ASFVPAM») under similar conditions were able to cause a lethal infection with a maximum temperature of 40.7±0.37°C and duration of fever on average 3.9±0.27 days (17 of 20 piglets). Low-virulent clones were revealed by direct immunofluorescence in pulmonary and spleen smears of clinically healthy piglets on days 14 and 17 post-infection (p.i.); their antigens were visualized in Vero cells by indirect immunoperoxidase method after 48 h p.i. at dose about 0.01 lg HAdU50/cm 3. They caused «crumbly» hemadsorption of infected Vero cells and their virions had typical for Asfarvirus view and size (210—220 nm). The obtained data served as the basis for analysis of the mechanism of rooting ASF agents in West Podillia enzootic foci, as well as for implication of the concept of low-budget quality control of anti-epizootic measures and evaluation of antiviral drugs’ activities against ASF. Conclusions. Low-cost operational procedures have been developed that allow one to use a vivarium of laboratory animals for ASF bioassay and meet principal requirements for science-based research in important aspects of experimental ASF epizootology. With their help, confirmation of the heterogeneity of the population of the ASF virus circulating in endemic foci of the Ukrainian Western Podillia was obtained (p&lt;0.05, n=25)&gt;&lt;0.05, n=25). The developed methodological approach is suitable for the study of fundamental issues of ASF epizootology, as well as for the quality control of anti-epizootic measures against ASF. In particular, it is advisable to use it to improve the biosecurity of agricultural export programs in Ukraine, a country that is disadvantaged by ASF.