Search Results (2,020)

Lumpy skin disease virus (LSDV), a Capripoxvirus genus member, causes severe cattle disease. Though Capripoxviruses share high nucleotide sequence homology indicating common ancestry, they have evolved distinct host adaptations. The LSDV genome encodes numerous proteins, with ORF133 being LSDV-specific and lacking clear homologs in other capripoxviruses, implying potential roles in host range and virulence. To explore ORF133’s function, this study generated mouse polyclonal antibodies against ORF133 and constructed the ORF133-deleted recombinant virus (LSDVΔORF133-EGFP) via homologous recombination with an EGFP reporter. Preliminary characterization showed that ORF133 deletion affects viral replication. This study provides critical tools and theoretical references for subsequent investigations into the functional mechanisms underlying ORF133 in LSDV. Full article

Potato virus Y (PVY) and potato leafroll virus (PLRV) are the most damaging viruses for potato production worldwide. Mixed infections not only result in additive detrimental effects on plant growth and tuber yield but also complicate the development of durable and broad-spectrum viral resistance. Heterologous protection against PVY can be achieved through the expression of the coat protein (CP) of lettuce mosaic virus (LMV) (CPLMV), conferring resistance via a capsid protein-mediated mechanism. On the other hand, we have previously demonstrated that transgenic lines expressing the PLRV ORF2 (RepPLRV) exhibit resistance to different PLRV isolates. In this study, potato transgenic lines of cv. Kennebec expressing CPLMV and RepPLRV were developed to confer dual virus resistance. Transgenic and non-transgenic control plants were molecularly and phenotypically characterized in greenhouse and field conditions. Across multiple growing seasons, two selected transgenic lines consistently displayed robust resistance to both major viruses, without exhibiting yield penalties or noticeable phenotypic alterations. These results constitute a significant advancement, demonstrating that dual resistance to PVY and PLRV can be achieved while preserving the original agronomic performance of the cultivar. This breakthrough not only contributes to long-term crop productivity but also provides a more sustainable strategy for managing viral diseases in potato production. Full article

Porcine epidemic diarrhea virus (PEDV) is a major cause of fatal diarrhea in piglets. The continuous emergence of new variants, driven by recombination and mutation, poses a persistent global threat to the swine industry, resulting in significant economic losses. Therefore, ongoing surveillance of PEDV evolution is critical. In this study, we isolated a novel PEDV strain, designated PEDV/FJLY202201, from experimental intestinal samples collected from a diarrheal piglet in Fujian, China, and sequenced its complete genome. Complete genome analysis, phylogenetic analysis, and recombination analysis were conducted. Results showed that PEDV/FJLY202201 was a recombinant strain derived from two recombination events between G2a and G2b strains, with three breakpoints located in the ORF1b, Domain 0 (D0) and S2 subunit, respectively. Notably, multiple mutations were identified in the S2 subunit, a finding that has been rarely reported before. Furthermore, following challenge with the PEDV/FJLY202201 strain, 3-day-old piglets exhibited severe diarrhea, sustained a 30.35% weight loss, and reached 100% mortality, collectively demonstrating its high virulence. These data reveal the complex evolution of PEDV/FJLY202201 and provide a foundation for a better understanding of the genetic evolution and molecular pathogenesis of PEDV. Full article

ORF8 is the second most mutated protein in SARS-CoV-2. It can form oligomers such as trimers and can bind to the IL-17RA/RC receptor. To understand the possible role of ORF8 in SARS-CoV-2, the first step of this study involved predicting the ORF8 trimer structure and the complex structure of the ORF8 monomer bound to the IL-17RA receptor using docking and molecular dynamics simulation methods. It was found that ORF8 molecules bound to the central ORF8 molecule through covalent and noncovalent interactions exhibit similar RMSD and RMSF values as the central ORF8 molecule and form a similar buried surface area, but display different numbers of hydrogen bonds and varying dynamic correlations. Additionally, trimer formation increases the dynamic correlation of the noncovalently bound ORF8 unit. ORF8 can bind with the IL-17RA receptor stably. Regions on ORF8, including C25–I47, L60–S67, T80–C90, and S103–E110, and regions on IL-17RA, including L1–H63 and D122–M165, are involved in the binding interface of the complex. ORF8 becomes less rigid when bound to IL-17RA than in its monomer, dimer, and trimer forms. Based on dihedral angle correlation predictions, binding of ORF8 to IL-17RA reduces internal correlations within ORF8 while strengthening correlations within IL-17RA. The G50–T80 region of ORF8 appears to be critical for interaction with IL-17RA, and the L1–V150 region of IL-17RA should be critical for its dynamics once bound to ORF8. These results help elucidate the structure and dynamics of ORF8 in SARS-CoV-2. Full article

Cells are continuously exposed to physiological and environmental stressors that disrupt homeostasis, triggering adaptive mechanisms such as the integrated stress response (ISR). A central feature of ISR is the selective translation of activating transcription factor 4 (ATF4), which orchestrates gene programs essential for metabolic adaptation and survival. Stress-induced acute ATF4 expression occurs in diverse mammalian cell types and is typically protective; however, chronic activation contributes to pathologies including cancer and neurodegeneration. Canonical ISR (c-ISR) is initiated by phosphorylation of eIF2α in response to stressors such as endoplasmic reticulum or mitochondrial dysfunction, hypoxia, nutrient deprivation, and infections. This modification suppresses global protein synthesis while promoting ATF4 translation through upstream open reading frames (uORFs) in its 5′UTR. Recently, an alternative pathway, split ISR (s-ISR), enabling ATF4 translation independently of eIF2α phosphorylation, was identified in mice, suggesting ISR adaptability, though its relevance in humans remains unclear. Under normal conditions, cap-dependent translation predominates, mediated by the eIF4F complex and requiring the activity of eIF2B at its initial steps. During translational stress, eIF2α phosphorylation inhibits eIF2B activity, resulting in the formation of stalled initiation complexes, which can aggregate into stress granules (SGs). SGs sequester mRNAs and translation initiation factors, further repressing global translation, while ATF4 mRNA largely escapes sequestration, enabling selective translation. This partitioning highlights a finely tuned regulatory mechanism balancing ATF4 expression during stress. Recent advances reveal that, beyond cis-regulatory uORFs, trans-acting factors such as translation initiation factors and associated RNA-binding proteins critically influence ATF4 translation. Understanding these mechanisms provides insight into ISR plasticity and its implications for development, aging, and disease. Full article

Although the SARS-CoV-2 pandemic no longer poses a global emergency, the virus continues to diversify and acquire immunoevasive properties. Understanding the molecular pathways that shape SARS-CoV-2 pathogenesis has become essential. In this paper, we summarize the most recent current evidence on how the spike protein structurally evolves, on changes in key non-structural proteins, such as nsp14, and on host factors, such as TMPRSS2 and neuropilin-1. These changes, together, shape viral entry, replication fidelity and interferon antagonism. Given the emerging Omicron variants of SARS-CoV-2, recent articles in the literature, cryo-EM analyses, and artificial intelligence-assisted mutational modeling were analyzed to infer and contextualize mutation-driven mechanisms. It is through these changes that the virus adapts and evolves, such as optimizing angiotensin-converting enzyme binding, modifying antigenic surfaces, and accumulating mutations that affect CD8⁺ T-cell recognition. Multi-omics data studies further support SARS-CoV-2 pathogenesis through convergent evidence linking viral adaptation to host immune and metabolic reprogramming, as occurs in myocarditis, liver injury, and acute kidney injury. By integrating proteomic, transcriptomic, and structural findings, this work presents how the virus persists and dictates disease severity through interferon antagonism (ORF6, ORF9b, and nsp1), adaptive immune evasion, and metabolic rewiring. All these insights underscore the need for next-generation interventions that provide a multidimensional framework for understanding the evolution of SARS-CoV-2 and guiding future antiviral strategies. Full article

Background: The enhanced sensitivity of next-generation sequencing (NGS) for assessing hepatitis B virus (HBV) quasispecies heterogeneity over clone-based sequencing (CBS) is well documented. However, its comparative reliability for genotype determination remains an open question. Objective: This study aimed to directly compare the performance of NGS and CBS for genotyping HBV using the entire viral genome. Methods: We selected five challenging clinical samples that previously could not be subgenotyped or showed conflicting results when using direct sequencing of the S open reading frame (ORF). The full HBV genome from these subjects was amplified and then analyzed in parallel by both NGS and CBS. Phylogenetic analysis was subsequently used to assign genotypes. Results: Both methods identified a range of genotypes, including B, C, and I, as well as aberrant and recombinant forms. For three of the five subjects, genotyping results were identical between the two platforms. In the remaining two cases, however, CBS revealed greater complexity, identifying additional subgenotypes and recombinant/aberrant strains not detected by NGS. Notably, for three individuals, the genotypes determined by both modern methods contradicted earlier results from 2011 based on direct S ORF sequencing. Furthermore, the specific mutations detected were incongruent between the platforms, with CBS identifying a higher number of variants than NGS. Conclusions: Our findings indicate that genotyping results from NGS and CBS can be discordant. Contrary to expectations, CBS may uncover more genetic diversity, including a greater number of subgenotypes and mutations, than NGS in certain contexts. The study also confirms that genotyping based solely on direct sequencing of the S ORF can be unreliable and lead to misclassification. Full article

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR showed that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we propose that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage. Full article

Clostridial neurotoxins, botulinum neurotoxins (BoNTs), and tetanus neurotoxin (TeNT) are potent toxins responsible for severe diseases, botulism and tetanus, respectively. BoNTs associate with non-toxic proteins (non-toxic non-hemagglutinin, hemagglutinins, and OrfXs), which protect BoNTs against acidic pH and protease degradation and facilitate BoNT passage through the intestinal barrier. TeNT enters motor neurons and undergoes a retrograde axonal transport until the target inhibitory interneurons in the central nervous system. BoNTs and TeNT recognize specific cell surface receptors which consist of complex sets of protein(s)-glycan-gangliosides and determine specific cell entry pathways. Recent data on structural and functional investigations of BoNT and TeNT receptors bring a better understanding of toxin trafficking in the host and entry into target neuronal cells, which is useful for the development of updated strategies of prevention and treatment of the corresponding diseases. Since clostridial neurotoxins, notably BoNTs, are important therapeutic tools, detailed knowledge of their activity opens the way of the development of engineered molecules for specific clinical applications. Full article

Disease-specific diversity in RNA transcripts stems from RNA splicing, ribosomal abnormalities, and other factors. However, the mechanisms underlying the regulation of rRNA expression in the nucleolus and mRNA expression in the cytoplasm during cancer and neuronal differentiation remain largely unknown. In this article, we review current knowledge and discuss the regulatory mechanisms of rRNA and mRNA expression in human diseases using the splicing model of PUF60 (poly(U) binding splicing factor 60)—also known as FUSE-binding protein-interacting repressor (FIR) (FUBP1-interacting repressor), RoBPI, SIAHBP1, and VRJS (Gene ID: 22827). Noncoding RNAs, much like coding RNAs, have been found to be translated into proteins with significant physiological functions. Splicing is also involved in dominant ORF RNAs implicated in the expression of both noncoding and coding RNAs. Here, we analyze recent findings regarding gene splicing, ribosome formation, and the determination of selected ORFs (dominant ORFs) in a system modeled on FIR splicing in two databases (RefSeq and ENSEMBL). rRNA transcription affects ribosomes, whereas mRNA expression and splicing affect the intracellular proteome. Our objective is to develop efficient methods for identifying biomarkers for disease diagnosis and therapeutic targets. In the field of cancer treatment, therapeutic drugs targeting intracellular signaling have proven effective. Full article

Cryptochromes (CRYs) are a conserved class of blue light and near-ultraviolet light receptors that regulate diverse processes, including photomorphogenesis in plants. In the extreme Antarctic environment, ice algae endure intense UV radiation, prolonged darkness, and low temperatures, where cryptochromes play a vital role in light sensing and stress response. In this study, we cloned the complete open reading frame (ORF) of the cryptochrome gene CiCRY-DASH1 from the Antarctic microalga Chlamydomonas sp. ICE-L. Both in vivo and in vitro DNA photorepair assays showed that CiCRY-DASH1 effectively repairs cyclobutane pyrimidine dimer (CPD) and 6-4 photoproducts (6-4PPs) induced by UV radiation. Furthermore, deletion of the N-terminal and C-terminal loop regions, combined with activity assays, revealed that the C-terminal loop region plays a crucial role in photorepair activity. These findings elucidate the adaptive photorepair mechanisms of Antarctic microalgae and establish CiCRY-DASH1 as a valuable genetic resource. Specifically, the high catalytic efficiency and evolutionary robustness of the engineered variants position it as a promising marine bioactive agent for photoprotective therapeutics and a strategic target for constructing microbial chassis to enable sustainable drug biomanufacturing. Full article

RhoA (Ras homolog A) is a prominent member of the Rho GTPase family, playing a key role in various cellular processes such as cytoskeletal dynamics, cell migration, and immune responses. However, its function in red swamp crayfish remains unclear. In this study, it is proposed that RhoA may regulate the innate immune response in P. clarkii. The gene was fully characterized as PcRhoA in P. clarkii. The results showed that the open reading frame (ORF) of PcRhoA is 663 bp, encoding a 220-amino acid protein with a conserved Rho domain of 174 amino acids. Phylogenetic analysis placed PcRhoA close to Cherax quadricarinatus RhoA. RT-qPCR analysis revealed high expression levels of the PcRhoA gene in the hepatopancreas, muscle, heart, ovary, and stomach, with lower expression in the blood, intestine, gills, and tentacle gland. Furthermore, PcRhoA mRNA transcript was significantly upregulated in the intestine following LPS and Poly I:C challenges. Knockdown of PcRhoA suppressed the expression of downstream genes in the immune signaling pathway. These results indicate that PcRhoA appears to play a pivotal role in regulating the immune response of crayfish. Full article

Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime–boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime–boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4⁺ and CD8⁺ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime–boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens. Full article

Ring Box Protein-1 (RBX1) is an essential component of the Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase, which is involved in the regulation of oocyte maturation in the form of ubiquitination substrate modification. In this study, a sequence of RBX1 (Sp-RBX1) was identified and analyzed using bioinformatics methods from the transcriptome data of Scylla paramamosain. The length of Sp-RBX1 cDNA sequence was 1247 bp, consisting of a 336 bp open reading frame (ORF). Sequence analysis revealed that the protein contained a C-terminal modified RING-H2 finger domain, with two zinc binding sites and a Cullin binding site, classifying it as a member of the RBX1 superfamily. The results of real-time fluorescence quantitative PCR (RT-qPCR) showed that Sp-RBX1 expression in the ovary was low at stages I and II, then significantly increased from stage III to V (p < 0.05), which indicated that it might be closely related to the maturation of oocytes. It also peaked at stage II in the hepatopancreas, then sharply declined from stages III to V. The expression pattern might be related to the accumulation of fat in the early development of hepatopancreas. Furthermore, we characterized the expression of Sp-RBX1 induced by follicle-stimulating hormone (FSH) and estradiol (E2) hormones. The results showed that the expression in the ovary was up-regulated by FSH and significantly inhibited by E2. The expression in the hepatopancreas increased only at 0.5 µmol/L concentration of FSH, and decreased in other groups. Conversely, it was up-regulated by E2. Thus, the expression of Sp-RBX1 was influenced by FSH in a concentration-dependent manner. These findings could offer valuable insights for further research on ovarian maturation in crustaceans. Full article

Zebrafish is emerging as a model animal for phenotype-based drug screening. Drugs screened from the zebrafish platform have advanced into clinical trials, underscoring their translational potential. Amyotrophic lateral sclerosis is a progressive motor neurons (MN) degenerative disease with few approved drugs. Previously, supplementation with exogenous recombinant phosphoglycerate kinase 1 (Pgk1) was found to improve MN growth through its interaction with receptor Eno2. To bypass the high complexity and cost of full-length Pgk1 production, a short segment within Pgk1 (M08) was predicted as the key motif interacting with Eno2, and a zebrafish phenotypic screening platform was established to find the most neurotrophic compound(s) among M08 and its mutants. We first found that M08-injected zebrafish embryos significantly increased branched caudal primary MNs (CaPMNs). However, compared to M08 (59.20 ± 1.80%), M039, among 17 mutants further screened, showed even more improvement of branched CaPMNs, up to 74.54 ± 3.73%. Next, when we administered the M039 peptide to C9ORF72-knockdown ALS-like zebrafish embryos, it improved axonal growth and swimming ability. Then, we employed a cellular model as a secondary screen, and M039 exhibited improved neurite outgrowth of MN (NOMN) and reduced p-Cofilin in NSC34 neural cells grown in ALS-like condition. Therefore, by using a zebrafish MN phenotype as a primary screening platform, we identified a mutated short peptide M039 having the most pronounced positive effect on improving neurite growth among all 17 mutants in comparison to parental M08, demonstrating the feasibility of zebrafish screening as a cost-effective strategy for finding promising neuroprotective short peptides that serve as neurotherapeutic potentials. Full article