Search Results (353)

Peste des petits ruminants virus (PPRV) has been classified into four lineages based on the nucleocapsid and fusion genes, with lineage IV strains being the most widely distributed. In Africa, recent epidemiological data revealed that PPRV lineage IV is increasingly displacing other lineages in prevalence, suggesting a competitive advantage in viral transmission and adaptability. Moreover, a lineage IV strain was the only confirmed strain in Europe and Asia. In this study, a one-step Taqman quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for lineage IV PPRV was established by targeting the hemagglutinin (H) gene. The results indicated that this method could detect approximately six copies of PPRV RNA, indicating high sensitivity. No cross-reactions with related viruses or other lineages of PPRV were observed. The results of a repeatability test indicated that the coefficient of variation values were low in both the inter-assay and intra-assay experimental groups. Detection of field samples indicated that all positive samples could be detected successfully using the developed method. This RT-qPCR assay provides a valuable tool to facilitate targeted surveillance and rapid differential diagnosis in regions with active circulation of PPRV lineage IV, enabling timely epidemiological investigations and strain-specific identification. Full article

The novel goose parvovirus (NGPV) and the novel duck reovirus (NDRV) are pathogens that can substantially affect the growth and development of ducklings, causing considerable economic losses to duck farms. Therefore, a timely, rapid, accurate, and high-throughput diagnosis and identification of viral infections are critical for preventing the spread of epidemics. In this study, a TaqMan probe-based duplex one-step RT-qPCR was established for the simultaneous detection and qualitative and quantitative identification of the two viruses. It demonstrated greater sensitivity than conventional PCR, detecting as low as 2.42 copies/μL of NGPV genome and 70.1 copies/μL of NDRV genome. Additionally, it exhibited remarkable specificity, responding exclusively to the nucleic acids of target pathogens. It also demonstrated excellent reproducibility and availability, particularly in clinical settings, with a coinfection detection rate of 13.3%, contributing to the development of NGPV- and NDRV-testing technologies. Full article

Feline coronavirus (FCoV), the causative agent behind feline infectious peritonitis (FIP), is one of the biggest infectious threats to feline health. Despite this threat, the tissue distribution and viral RNA levels in cats infected with feline coronaviruses are poorly understood in the context of natural infection. Here, we used a two-step reverse-transcription quantitative PCR (RT-qPCR) to examine viral RNA levels from different sampling sites in both cats that have been clinically suspected of FIP and their feline housemates. We show that the distribution and amount of FCoV viral RNA does not differ between FCoV-infected cats with FIP and their feline housemates in blood, conjunctiva, or feces. Furthermore, in all FIP and non-FIP cases, viral RNA levels were higher in fecal samples than the blood. Taken together, these results show that amount of viral RNA does not differ between FCoV-infected cats with FIP and their healthy housemates in several sample types. Our results indicate a need for closer examination of FCoV pathogenesis independent of viral dissemination, including an assessment of intrahost evolution of FCoVs and FCoVs’ interactions with the feline immune system. Full article

MicroRNAs are key regulators of lymphoid differentiation, exhibiting a pivotal role in acute lymphoblastic leukemia (ALL) biology and prognosis. The initial steps of canonical miRNA biogenesis involve the microprocessor complex processing the primary miRNA transcripts into precursor miRNAs via Drosha. DROSHA polymorphisms have been implicated in pediatric ALL and linked with cancer risk. This study investigated the role of rs642321, rs3805500, and rs10035440 DROSHA polymorphisms in ALL susceptibility, relapse, and outcomes in children and adolescents of Greek descent. The study included 252 children and adolescents (115 ALL cases and 137 controls). Genotyping was performed using RT-qPCR and the TaqMan Genotyping Assay. Homozygotes for the minor allele in DROSHA rs642321 were nominally associated with ALL susceptibility (TT vs. CC+CT; OR 4.5; 95% CI: 1.2–21.2; p_adj = 0.034). Likewise, homozygotes for the minor allele in rs3805500 were linked with ALL risk (GG vs. AA+AG; OR 2.7; 95% CI: 1.3–6.1; p_adj = 0.012). A suggestive association was observed between the rs3805500 AG genotype and both relapsed (OR 5.8; 95% CI: 1.6–24.3; p_adj = 0.011) and deceased cases (OR 5; 95% CI: 1.1–26.3; p_adj = 0.038). Patients with the rs3805500 AG and GG genotypes showed a trend toward poorer overall survival rates. In summary, certain haplotypes of DROSHA polymorphisms may be modestly associated with the occurrence of childhood ALL and its outcomes, although these findings require validation in larger, independent cohorts. Full article

Background: Hypertrophy, myogenic differentiation, and mass gain of porcine skeletal muscle are key factors in meat production efficiency, regulated by miRNAs through post-transcriptional mechanisms. This study aims to identify miRNA-mRNA pairs linked to growth and muscle development in Jiangquan Black pigs with differing average daily gains (ADGs), providing a foundation for molecular breeding in this breed. Methods: This study divided eight pigs into two groups and analyzed the skeletal muscle characteristics of Jiangquan Black pigs with different average daily weight gains using HE staining. RNA-Seq was conducted to identify differentially expressed miRNAs and mRNAs, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and an integrated miRNA-mRNA regulatory network was subsequently constructed. Results: RNA sequencing analysis identified 255 differentially expressed genes (DEmRNAs, |FC| > 1.5) and 27 differentially expressed miRNAs (DE miRNAs, |FC| > 2). Bioinformatics analysis revealed 330 significantly negatively correlated miRNA-mRNA regulatory pairs, with key pathways, including the MAPK, mTOR, insulin, FoxO, Wnt, and TGF-β signaling pathways, being implicated in muscular development. Quantitative real-time PCR (qRT-PCR) validation confirmed the reliability of the sequencing data. Conclusions: Different ADGs among half-sibling Jiangquan Black pigs with the same diet may be due to the DE miRNAs and DEmRNAs related to skeletal muscle growth and development. These findings reveal the potential regulatory mechanisms of DE miRNAs and DEmRNAs in porcine skeletal muscle growth, providing valuable insights for the next steps in molecular breeding strategies for Jiangquan Black pigs. Full article

Bougainvillea spp. possesses vibrantly pigmented bracts that exhibit high ornamental value. Zinc finger proteins (ZFPs), one of the most extensive transcription factor families in plants, are implicated in diverse biological functions, including plant morphogenesis, transcriptional regulation, and responses to abiotic stress. Nevertheless, their regulatory roles in bract pigmentation in Bougainvillea remain unexplored. In the present investigation, 105 BbZFP genes were identified from the Bougainvillea genome via bioinformatic analyses and subsequently categorized into five subgroups according to the quantity and arrangement of their structural domains. Analysis of physicochemical characteristics demonstrated that the BbZFP family encompasses both acidic and basic proteins, all of which are hydrophilic and predominantly classified as unstable proteins. Gene structure analysis revealed that the majority of BbZFP genes comprise between one and five– introns. Cis-regulatory element analysis suggested that BbZFP promoter regions harbor multiple elements associated with abiotic stress responses, hormonal regulation, and light responsiveness, implying their possible participation in these physiological processes. Transcriptomic data analysis revealed distinct expression patterns of BbZFP genes among bracts of different colors. A quantitative real-time polymerase chain reaction (RT-qPCR) further confirmed that Bou_68928, Bou_1096, Bou_4400, and Bou_17631 were markedly upregulated in yellow bracts relative to white bracts, suggesting their involvement in flavonoid biosynthesis regulation. Meanwhile, Bou_1096 and Bou_17631 exhibited markedly elevated expression in red-purple bracts compared to white bracts, potentially regulating betacyanin biosynthesis in Bougainvillea. These findings offer candidate genes for molecular breeding strategies aimed at enhancing floral coloration in Bougainvillea. The next step will involve elucidating the functions of these genes in bract coloration. Full article

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the zoonotic virus responsible for the COVID-19 pandemic, has caused global health and economic disruption. American mink (Neovison vison) are highly susceptible to SARS-CoV-2 and capable of transmitting it to both mink and humans. We previously reported the first detection of SARS-CoV-2 in feral mink, with two positive cases among 13 animals in the upper courses of two rivers in the Valencian Community, eastern Spain. Here, we expand that study with 60 additional feral mink sampled from November 2020 to May 2022. Four new positives were identified by two-step RT-PCR assay on necropsy samples, including nasal and rectal swabs, lung tissue, lymph nodes, and fetuses from three pregnant females. In total, six of 73 mink tested positive, all with low viral loads. Sanger sequencing confirmed infection and revealed clustering with the B.1.177 and Alpha variants. Body weight and reproductive status analyses indicated seasonal breeding and high population turnover, consistent with other wild mink populations. Our findings reveal that SARS-CoV-2 circulation is limited in feral mink, at least in this region. They underscore the key importance of wildlife surveillance as an element of the One Health strategy, which encompasses humans, animals, and the environment. Full article

Background/Objectives: The COVID-19 pandemic, caused by SARS-CoV-2, required the rapid development of diagnostic tests. SARS-CoV-2, part of the betacoronavirus genus, shares characteristics with SARS-CoV-1, including its ability to survive on surfaces, facilitating the spread of the infection. This study analyzes the technique of nasopharyngeal secretion collection for SARS-CoV-2 diagnosis and compares the accuracy of rapid antigen and molecular tests. Methods: This study had two components: study A assessed the healthcare personnel training in collecting nasopharyngeal secretions and the discomfort associated with applying a questionnaire. Study B compared rapid antigen test accuracy with RT-PCR among children, through a retrospective analysis. The data were statistically analyzed to assess compliance with the testing protocols. Results: In study A, 88 healthcare workers achieved an average compliance score of 7.60 out of 10 regarding the collection procedure. Over 70% of participants correctly followed the fundamental steps of the procedure. Many patients who underwent sample collection reported pain and symptoms such as coughing or sneezing. In study B, 198 pediatric patients were tested using rapid antigen tests, collected simultaneously with RT-PCR. The rapid tests showed a 50% sensitivity and 97.5% specificity. Conclusions: This study indicates that nasopharyngeal specimen collection techniques are based on international recommendations, but improvements could be made to reduce discomfort. Rapid antigen tests are helpful for screening due to their high specificity and negative predictive value. Continuous healthcare personnel training and the monitoring of diagnostic techniques remain essential in managing SARS-CoV-2 and other viral infections. Full article

Background/Objectives: Achromobacter xylosoxidans and Achromobacter insuavis are emerging opportunistic pathogens. Several Resistance-Nodulation-cell Division (RND) efflux systems are involved in intrinsic or acquired antibiotic resistance (AxyABM, AxyXY-OprZ, and AxyEF-OprN). The aim of this study was to explore the resistance mechanisms in one-step mutants in which the efflux systems described to date are not involved: one mutant of A. insuavis AXX-A (AXX-A-Do1) and two mutants of A. xylosoxidans CIP102236 (CIP102236-El9 and CIP102236-Eo4) selected on fluoroquinolones. Methods: In vitro mutants were compared to parental isolates by WGS. RT–qPCR and gene inactivation were used to explore the role of the new efflux systems detected. Results: In the A. insuavis AXX-A mutant (AXX-A-Do1), WGS showed a substitution in the putative regulator of the new RND efflux system AinCDJ. The transporter gene ainD was 79-fold overexpressed in AXX-A-Do1, compared to its parental strain. The inactivation of ainD in AXX-A-Do1 led to a decrease in MICs of ciprofloxacin (8-fold), levofloxacin (8-fold), cefepime (≥8-fold), meropenem (4-fold), doripenem (4-fold), doxycycline (4-fold), minocycline (4-fold), tigecycline (4-fold) and chloramphenicol (≥8-fold). The MICs values obtained were similar to those of the parental strain AXX-A. The same approach allowed the detection of the new efflux system AxySUV in A. xylosoxidans CIP102236 mutants, in which substitutions in the putative AxySUV regulator were associated with the overexpression of the transporter gene axyU. axyU inactivation in the mutants led to a decrease in MICs of ciprofloxacin (8- to 16-fold), levofloxacin (4- to 8-fold), doripenem (4-fold), doxycycline (4-fold), minocycline (4-fold), and chloramphenicol (≥4-fold). Interestingly, axySUV is present in only about 50% of available A. xylosoxidans genomes, whereas ainCDJ is detected in all A. insuavis genomes. Conclusions: This study demonstrated that AinCDJ overproduction is involved in the acquired resistance of A. insuavis to cefepime, meropenem, doripenem, fluoroquinolones, minocycline, doxycycline, tigecycline, and chloramphenicol and that AxySUV overproduction is involved in the acquired resistance of A. xylosoxidans to meropenem, fluoroquinolones, minocycline, doxycycline, and chloramphenicol. Full article

Porcine transmissible gastroenteritis virus (TGEV) is a highly contagious pathogen causing severe diarrhea in pigs, particularly piglets, leading to significant economic losses. Distinguishing TGEV from the genetically similar porcine respiratory coronavirus (PRCV) remains challenging due to their high genomic homology. In this study, we developed a one–pot assay combining recombinase-aided amplification (RAA) and CRISPR/Cas13a technology, targeting the TGEV S gene. This method was optimized for sensitivity and specificity, with orthogonal tests determining the optimal reagent concentrations. The assay achieved a detection limit of 4.13 copies/µL within 40 min at 37 °C, demonstrating no cross-reactivity with other porcine viruses. Clinical validation on 140 samples showed 100% concordance with RT–qPCR and RT–PCR results. Since the established method is completed in a single reaction tube, it eliminates the need for step-by-step operations, simplifying the process and reducing the risk of cross–contamination and false positives in subsequent tests. Overall, this assay shows promising potential for TGEV detection. Full article

Background: Acyl-CoA oxidase (ACX), a ubiquitous eukaryotic enzyme, catalyzes the initial steps of fatty acid β oxidation and plays an important role in the biosynthesis of jasmonic acid (JA). At present, no studies have been reported on ACX family members of maize and their function in disease resistance. Objectives: This study aims to lay a foundation for clarifying the functions of ACX family genes in maize growth, development, and stress response by conducting a genome-wide identification of ACX family genes in maize, analyzing the expression characteristics of these genes in maize growth and development, hormone treatment and response to biotic and abiotic stresses, and exploring the functions of key genes in the maize disease resistance process through the use of mutants. Methods: ProtParam, TBtools, MEME, MEGA, and IBS tools were used to identify maize ACX family genes and analyze the physicochemical properties of their proteins, chromosome location, phylogenetic relationships among family members, conserved domains, conserved motifs, and cis-acting elements. Meanwhile, the expression patterns of maize ACX family genes in different tissues and their expression patterns under abiotic and biotic stresses were studied by using the data from the maize GDB database and qRT-PCR technology. Moreover, the mutants of ZmACX1, ZmACX3, ZmACX4, and ZmACX5 genes were obtained, and the disease resistance of the mutants was detected to further determine the functions of ACX genes in the maize disease resistance process. This study identified maize ACX family genes using bioinformatics methods. Results: We discovered that six ACX genes in the maize genome are distributed across four different chromosomes. Cluster analysis further classified these genes into three subfamilies. All maize ACX genes possess a conserved ACOX domain, and their promoter regions are enriched with cis-acting elements associated with heat stress and the plant hormone response. Under various tissue, biotic, and abiotic stress conditions, as well as treatments with methyl jasmonate (MeJA) and salicylic acid (SA), the expression levels of maize ACX family genes exhibited significant differences. Notably, the expression levels of ZmACX1, ZmACX3, ZmACX4, and ZmACX5 were significantly up-regulated following stress and pathogen infection, suggesting their involvement in maize growth, development, and disease resistance. To elucidate the function of these genes in maize disease resistance, the resistance of ZmACX1, ZmACX3, ZmACX4, and ZmACX5 mutants to Cochliobolus heterostrophus, Curvularia lunata, and Fusarium graminearum were further examined. The results revealed that compared to the wild-type B73, the lesion area of the mutants was significantly increased after inoculation with pathogens. This directly demonstrated the crucial role of these genes in maize resistance to C. heterostrophus, C. lunata, and F. graminearum. Conclusions: In summary, this study systematically identified maize ACX family genes, and thoroughly investigated their expression patterns and functions in maize disease resistance. Our findings provide valuable insights into the comprehensive understanding of the function and mechanism of maize ACX family genes. Full article

Background: Diabetes mellitus (DM) is a common potentially life-threatening endocrine disorder in pets and humans. Since only symptomatic treatment is available, a more sustainable treatment is urgently needed. Objective: The aim of this study is to establish functional differentiated canine pancreatic β-cells that release insulin upon glucose stimulus. Methods: Pancreatic tissue was obtained from surplus material of healthy dogs (n = 4), euthanized for non-pancreatic related research. Ductal cells were isolated and expanded in dog pancreas expansion media (dpEM) and differentiated and maturated in five sequentially added pancreas differentiation media (PDMs). Gene expression was analyzed by reversed transcriptase qPCR (RT-qPCR), and insulin release was analyzed with a canine-specific ELISA. Results: Canine pancreatic ductal cells (LGR5 and SOX9 expression) were differentiated into β-cells expressing key β-cell-related genes: Pancreatic and duodenal homeobox 1 (PDX1), NK6 Homeobox 1 (NKX6.1), Glucose Transporter Type 2 (GLUT2), Proprotein convertase subtilisin/kexin type 1 (PCSK1), and low levels of insulin. Neither Glucagon (α-cells) nor LGR5 and SOX9 were expressed, and somatostatin was expressed at low levels. The differentiated cells released insulin upon glucose stimulation. Conclusion and implications: The step-by-step differentiation protocol, mimicking pancreatic organogenesis, resulted in β-cells secreting insulin levels suitable for β-cell disease modelling. It remains to be seen if stem cells from diseased animals behave similarly. Full article

MicroRNAs (miRNAs), which circulate in the serum and plasma, play a role in several biological processes, and their levels in body fluids are associated with the pathogenesis of various diseases, including different types of cancer. For this reason, miRNAs are considered promising candidates as biomarkers for diagnostic purposes, enabling the early detection of pathological onset and monitoring drug responses during therapy. However, current methods for miRNA quantification, such as northern blotting, isothermal amplification, RT-PCR, microarrays, and next-generation sequencing, are limited by their reliance on centralized laboratories, high costs, and the need for specialized personnel. Consequently, the development of sensitive, simple, and one-step analytical techniques for miRNA detection is highly desirable, particularly given the importance of early diagnosis and prompt treatment in cases of cancer. Lateral flow assays (LFAs) are among the most attractive point-of-care (POC) devices for healthcare applications. These systems allow for the rapid and straightforward detection of analytes using low-cost setups that are accessible to a wide audience. This review focuses on LFA-based methods for detecting and quantifying miRNAs associated with the diagnosis of various cancers, with particular emphasis on sensitivity enhancements achieved through the application of different labels and detection systems. Early, non-invasive detection of these diseases through the quantification of tailored biomarkers can significantly reduce mortality, improve survival rates, and lower treatment costs. Full article

The regulation of downstream responsive genes by transcription factors (TFs) is a critical step in the stress response system of plants. While bZIP transcription factors are known to play important roles in stress reactions, their functional characterization in soybeans remains limited. Here, we identified a soybean bZIP gene, GmbZIP60, which encodes a protein containing a typical bZIP domain with a basic region and a leucine zipper region. Subcellular localization studies confirmed that GmbZIP60 is localized in the nucleus. Expression analysis demonstrated that GmbZIP60 is induced by salt stress, drought stress, and various plant hormone treatments, including abscisic acid (ABA), ethylene (ETH), and methyl jasmonate acid (MeJA). Overexpressing GmbZIP60 (OE-GmbZIP60) in transgenic soybean and rice enhanced tolerance to both salt and drought stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression levels of abiotic stress-responsive genes were significantly higher in transgenic plants than in wild-type (WT) plants under stress conditions. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis further confirmed that GmbZIP60 directly binds to the promoters of abiotic stress-related genes induced by ABA, ETH, JA, and salicylic acid (SA). Overall, these findings revealed GmbZIP60 as a positive regulator of salt and drought stress tolerance. Full article

Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5′ untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28–1.58% and the inter-assay CV of 0.20–1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses. Full article