HuR-positive stress granules: Potential targets for age-related osteoporosis.

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target ß-catenin by binding and recruiting ß-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of ß-catenin into HuR-positive SGs, facilitated ß-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.

Genetic Diversity of Domestic Cat Hepadnavirus in Southern Taiwan.

Domestic cat hepadnavirus (DCH) is an infectious disease associated with chronic hepatitis in cats, which suggests a similarity with hepatitis B virus infections in humans. Since its first identification in Australia in 2018, DCH has been reported in several countries with varying prevalence rates, but its presence in Taiwan has yet to be investigated. In this study, we aimed to identify the presence and genetic diversity of DCH infections in Taiwan. Among the 71 samples tested, eight (11.27%) were positive for DCH. Of these positive cases, three cats had elevated levels of alanine transaminase (ALT) and aspartate transaminase (AST), suggesting an association between DCH infection and chronic hepatitis. Four DCH-positive samples were also tested for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) coinfection. One sample (25%) was positive for FIV, whereas there was no positive sample for FeLV (0%). In addition, we performed whole genome sequencing on six samples to determine the viral genome sequences. Phylogenetic analyses identified a distinct lineage compared with previously reported sequences. This study highlights the importance of continuous surveillance of DCH and further research to elucidate the pathophysiology and transmission route of DCH.

Research progress in biological control of tomato bacterial wilt.

Bacterial wilt caused by the infection of Ralstonia solanacearum, is one of the most harmful diseases to tomatoes, one of the most important greenhouse vegetables in China. R. solanacearum can survive and remain active in the deep soil for a long time, and the chemical control of tomato bacterial wilt is consequently limited. In this study, we introduced the characteristics of tomato bacterial wilt disease and the types of R. solanacearum, and systematically reviewed the research progresses of biological control methods from the aspects of botanical insecticides, agricultural antibiotics, biocontrol bacteria. We emphatically introduced the principle and current status of these methods, discussed the limitations and the improvement strategies, and prospected a new environmental protection and efficient biological control system based on micro-ecological regulation would be the development direction of biological control of tomato bacterial wilt.

Nitrous oxide emissions from tea plantations: A review.

Tea plantations are an important N2O source. Fertilizer-induced N2O emission factors of tea plantations are much higher than other upland agricultural ecosystems. According to the basic information on characteristics and knowledge of N2O emissions from tea plantations around the world, we comprehensively reviewed N2O emission characteristics, production process, influencing factors, and reduction measures from tea plantations. The global means of ambient N2O emission and N2O emission stimulated by nitrogen fertilizer application from tea plantations were (2.68±2.92) kg N·hm-2 and (11.29±9.45) kg N·hm-2, respectively. The fertilizer-induced N2O emission factor in tea plantations (2.2%±2.1%) was much higher than the IPCC-estimated N2O emission factor for agricultural land (1%). N2O emission from tea plantation soil (a typical acid soil) were mainly produced during nitrification and denitrification, with denitrification being dominant. N2O emission from tea plantations were significantly related to the amount of fertilizer application. Other factors, such as fertilizer type, could also affect soil N2O emissions in tea plantations. The main reduction methods of N2O emission from tea plantations included optimizing the amount and type of fertilizer, amending biochar, and rationally using nitrification inhibitors. In future, we should strengthen in-situ observations of soil N2O emission from tea plantations at both temporal and spatial scales, combine lab incubation and field studies to elucidate the mechanisms underling tea plantation soil N2O emissions, and use a data-model fusion approach to reduce uncertainties in the estimation of global N2O emission. These would provide theoretical support and practical guidance for reasonable N2O emission reduction in tea plantations.

MiR-12200-5p Targets Multiple Members of Wnt Signaling Pathway to Inhibit Osteoblast Differentiation and Bone Formation.

BACKGROUND: Osteoporosis is widespread and has become an emerging problem in the elderly. MicroRNAs could affect osteoblast differentiation and further regulate the occurrence of osteoporosis by targeting osteogenic differentiation signaling pathways. Our screening study found that miR-12200-5p simultaneously targeted six important factors within the Wnt signaling pathway (Apc, Tcf4, Tcf7, Wnt3a, Wnt5a, and Lrp6), indicating that miR-12200-5p might function as a strong regulator of this pathway. Since the Wnt pathway exists as one of the most essential pathways for osteogenic differentiation, miR-12200-5p may have an important role in the development of osteoporosis. OBJECTIVE: This study intended to explore the regulatory role and corresponding mechanism of miR-12200-5p in osteoblast differentiation. METHODS: We investigated the differentiation of osteoblast after the treatments of miR-12200-5p mimic and inhibitor. The interactions between miR-12200-5p and its target genes were also detected. Furthermore, the rescue effect of miR-12200-5p inhibitor on osteoporosis was evaluated using an ovariectomized osteoporosis mouse model. RESULTS: MiR-12200-5p significantly inhibited osteoblast differentiation, and bound with the 3'-UTR sequences of its target genes (Apc, Tcf4, Tcf7, Wnt3a, Wnt5a, and Lrp6) to reduce the expressions of these genes. The inhibition of miR-12200-5p would almost fully alleviate postmenopausal osteoporosis. CONCLUSION: MiR-12200-5p could strongly repress osteoblast differentiation and bone formation by targeting multiple members of the Wnt signaling pathway simultaneously. The study supplemented the theoretical and experimental basis for researching the mechanism of osteogenic differentiation and inspired the development of novel therapeutic strategies for osteoporosis.

How do RNA binding proteins trigger liquid-liquid phase separation in human health and diseases?

RNA-binding proteins (RBPs) lie at the center of post-transcriptional regulation and protein synthesis, adding complexity to RNA life cycle. RBPs also participate in the formation of membrane-less organelles (MLOs) via undergoing liquid-liquid phase separation (LLPS), which underlies the formation of MLOs in eukaryotic cells. RBPs-triggered LLPS mainly relies on the interaction between their RNA recognition motifs (RRMs) and capped mRNA transcripts and the heterotypic multivalent interactions between their intrinsically disordered regions (IDRs) or prion-like domains (PLDs). In turn, the aggregations of RBPs are also dependent on the process of LLPS. RBPs-driven LLPS is involved in many intracellular processes (regulation of translation, mRNA storage and stabilization and cell signaling) and serves as the heart of cellular physiology and pathology. Thus, it is essential to comprehend the potential roles and investigate the internal mechanism of RPBs-triggered LLPS. In this review, we primarily expound on our current understanding of RBPs and they-triggered LLPS and summarize their physiological and pathological functions. Furthermore, we also summarize the potential roles of RBPs-triggered LLPS as novel therapeutic mechanism for human diseases. This review will help understand the mechanisms underlying LLPS and downstream regulation of RBPs and provide insights into the pathogenesis and therapy of complex diseases.

A lipid-polymer hybrid nanoparticle (LPN)-loaded dissolving microneedle patch for promoting hair regrowth by transdermal miR-218 delivery.

Alopecia is the most common multifactorial hair loss disorder, affecting almost 50% of the population and even having a serious psychological impact on the patients. miR-218 has therapeutic potential for alopecia since it can activate the Wnt/ß-catenin channel by down-regulating SFRP2, which is a key channel in hair follicle cycle transformation for hair regrowth. Although miR-218 has the potential to treat this disease, several barrier properties of the skin challenge miRNA's delivery to the target location, such as passing through the corneum and resistant enzymatic degradation. To address these challenges, we evaluated a device that combined the use of hyaluronic acid (HA)-based dissolving microneedle (MN) to enhance corneum permeability with the lipid polymer hybrid nanoparticles (LPNs) as a miRNA delivery carrier to protect miR-218 from degradation. The MN patches could promote LPNs/miR-218 diffusing in the dermis region, and significantly increase the bioavailability of miR-218. Furthermore, in the shaved mouse model, the MN patches showed higher efficacy in promoting hair growth than the topical smear treatment, while avoiding the safety concern. This work established a novel and effective combination device with MN and LPNs that can be used for localized transdermal miRNA delivery to promote hair regrowth.

Liquid-Liquid Phase Separation of Biomacromolecules and Its Roles in Metabolic Diseases.

Liquid-liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into liquid-like condensates, which underlies membraneless organelles (MLOs) formation in eukaryotic cells. With increasing evidence of the LLPS concept and methods, this phenomenon as a novel principle accounts for explaining the precise spatial and temporal regulation of cellular functions. Moreover, the phenomenon that LLPS tends to concentrate proteins is often accompanied by several abnormal signals for human diseases. It is reported that multiple metabolic diseases are strongly associated with the deposition of insoluble proteinaceous aggregating termed amyloids. At present, recent studies have observed the roles of LLPS in several metabolic diseases, including type 2 diabetes mellitus (T2DM), Alzheimer's disease (AD), and metabolic bone diseases (MBDs). This review aims to expound on the current concept and methods of LLPS and summarize its vital roles in T2DM, AD, and MBDs, uncover novel mechanisms of these metabolic diseases, and thus provide powerful potential therapeutic strategies and targets for ameliorating these metabolic diseases.

Comprehensive ceRNA network for MACF1 regulates osteoblast proliferation.

BACKGROUND: Previous studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC­E1 pre­osteoblast cells. RESULTS: In total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes. CONCLUSION: This study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC­E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.

The Smad3-dependent microRNA let-7i-5p promoted renal fibrosis in mice with unilateral ureteral obstruction.

Renal fibrosis is a common feature of all types of chronic kidney disease (CKD) and is tightly regulated by the TGF-ß/Smad3 pathway. Let-7i-5p belongs to the let-7 microRNA family with diverse biological functions. It has been reported that let-7i-5p suppresses fibrotic disease in the heart, lungs, and blood vessels, while the role of let-7i-5p in renal fibrosis remains limited. In this study, we aimed to investigate the role of let-7i-5p in renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and TGF-ß1-stimulated renal tubular cell line TCMK1. The RNA-targeting CRISPR/Cas13d system was used to knock down let-7i-5p. Renal injury and fibrosis were determined by histological analysis, RT-PCR, Western blot, and immunostaining. Our results have shown that in the kidneys after UUO, the expression of let-7i-5p was significantly increased along with notable tubular injury and interstitial fibrosis. Electroporation of let-7i-targeting Cas13d plasmid efficiently knocked down let-7i-5p in kidneys after UUO with reduced tubular injury, fibrotic area, and expression of fibrotic marker genes α-SMA, fibronectin, and Col1a1. In TGF-ß1-stimulated TCMK1 cells, knockdown of let-7i-5p by Cas13d plasmid transfection also blunted the expression of fibrotic marker genes. Most importantly, the genomic locus of let-7i showed enriched binding of Smad3 as revealed by chromatin immunoprecipitation. In TCMK1 cells, the overexpression of Smad3 can directly induce the expression of let-7i-5p. However, the deletion of Smad3 abolished TGF-ß1-stimulated let-7i-5p expression. Collectively, these findings suggest that let-7i-5p is a Smad3-dependent microRNA that plays a pathogenic role in renal fibrosis. Let-7i-5p could be a promising target for the treatment of CKD-associated renal fibrosis.

MiR-138-5p Targets MACF1 to Aggravate Aging-related Bone Loss.

Senile osteoporosis is one of the major health problems in an aging society. Decreased bone formation due to osteoblast dysfunction may be one of the causes of aging-related bone loss. With increasing evidence suggesting that multiple microRNAs (miRNAs) play important roles in osteoblast function, the relationship between miRNAs and senile osteoporosis has become a popular research topic. Previously, we confirmed that mechanoresponsive miR-138-5p negatively regulated bone anabolic action. In this study, the miR-138-5p level was found to be negatively correlated with BMD and osteogenic markers in bone specimens of senile osteoporotic patients by bioinformatic analysis and experimental verification. Furthermore, high miR-138-5p levels aggravated the decrease of aged osteoblast differentiation in vitro and led to worse bone loss in aged osteoblastic miR-138-5p transgenic mice in vivo. We also previously identified that the target of miR-138-5p, microtubule actin cross-linking factor 1 (MACF1), could attenuate senile osteoporosis. Here, miR-138-5p was demonstrated to regulate aged osteoblast differentiation by targeting MACF1. Finally, the therapeutic inhibition of miR-138-5p counteracted the decrease in bone formation and aging-related bone loss in aged mice. Overall, our results highlight the crucial roles and the molecular mechanism of miR-138-5p in aging-related bone loss and may provide a powerful therapeutic target for ameliorating senile osteoporosis.

Calycosin prevents bone loss induced by hindlimb unloading.

Bone loss induced by microgravity exposure seriously endangers the astronauts' health, but its countermeasures still have certain limitations. The study aims to find potential protective drugs for the prevention of the microgravity-induced bone loss. Here, we utilized the network pharmacology approach to discover a natural compound calycosin by constructing the compound-target interaction network and analyzing the topological characteristics of the network. Furthermore, the hind limb unloading (HLU) rats' model was conducted to investigate the potential effects of calycosin in the prevention of bone loss induced by microgravity. The results indicated that calycosin treatment group significantly increased the bone mineral density (BMD), ameliorated the microstructure of femoral trabecular bone, the thickness of cortical bone and the biomechanical properties of the bone in rats, compared that in the HLU group. The analysis of bone turnover markers in serum showed that both the bone formation markers and bone resorption markers decreased after calycosin treatment. Moreover, we found that bone remodeling-related cytokines in serum including IFN-γ, IL-6, IL-8, IL-12, IL-4, IL-10 and TNF-α were partly recovered after calycosin treatment compared with HLU group. In conclusion, calycosin partly recovered hind limb unloading-induced bone loss through the regulation of bone remodeling. These results provided the evidence that calycosin might play an important role in maintaining bone mass in HLU rats, indicating its promising application in the treatment of bone loss induced by microgravity.

Immunity, Ion Channels and Epilepsy.

Epilepsy is a common chronic neurological disorder in modern society. One of the major unmet challenges is that current antiseizure medications are basically not disease-modifying. Among the multifaceted etiologies of epilepsy, the role of the immune system has attracted considerable attention in recent years. It is known that both innate and adaptive immunity can be activated in response to insults to the central nervous system, leading to seizures. Moreover, the interaction between ion channels, which have a well-established role in epileptogenesis and epilepsy, and the immune system is complex and is being actively investigated. Some examples, including the interaction between ion channels and mTOR pathways, will be discussed in this paper. Furthermore, there has been substantial progress in our understanding of the pathophysiology of epilepsy associated with autoimmune encephalitis, and numerous neural-specific autoantibodies have been found and documented. Early recognition of immune-mediated epilepsy is important, especially in cases of pharmacoresistant epilepsy and in the presence of signs of autoimmune encephalitis, as early intervention with immunotherapy shows promise.

Bergamottin promotes osteoblast differentiation and bone formation via activating the Wnt/ß-catenin signaling pathway.

Osteoporosis is one of the most common bone disorders that seriously affect the health and life quality of elderly individuals. Reduced osteoblast differentiation and bone formation lead to changes in bone volume and microarchitecture, leaving the bones vulnerable to fracture. Bergamottin (BM) is a natural compound derived from various citrus fruits and possesses multiple biological activities including anti-adipogenesis function. This study aimed to evaluate the effects of BM on osteoblast differentiation and its potential anti-osteoporosis capacity, as well as to explore the underlying mechanism. We demonstrated that BM, as a positive regulator for osteogenesis, significantly promoted osteoblast differentiation and bone formation. Mechanically, BM activated the Wnt/ß-catenin signaling pathway and promoted the nuclear translocation of ß-catenin. In addition, BM dramatically upregulated the expression of ß-catenin, enhanced the transcriptional activation of T cell factor 7 (TCF7), and increased the expression of Runt-related transcription factor 2 (Runx2). Taken together, this study revealed that BM enhanced osteoblast differentiation and attenuated ovariectomy (OVX)-induced bone loss, possessing the potential to be developed into a food ingredient or supplement for preventing osteoporosis.

Kaempferide enhances antioxidant capacity to promote osteogenesis through FoxO1/ß-catenin signaling pathway.

BACKGROUND: Forkhead box O1 (FoxO1)/ß-catenin signaling pathway is a main oxidative defense pathway, which plays essential roles in the regulation of osteoporosis (OP). The natural products possess quality therapeutic effects and few side effects. It is used as a novel strategy in the treatment of OP. However, there is no systematic study in the natural antioxidant drug based on the FoxO1/ß-catenin signaling pathway. This paper aims to discover pro-osteogenesis natural antioxidants for the prevention and treatment of OP. METHODS: Systems pharmacology; combined with reverse drug targeting, systems-ADME process, network analysis and molecular docking, was used to screen natural antioxidants based on the FoxO1/ß-catenin signaling pathway. Then in vitro experiments were performed to evaluate the osteogenesis effects of screened natural antioxidants. RESULTS: Kaempferide was screened as the most potential antioxidant to improve osteogenesis by the regulation of the FoxO1/ß-catenin signaling pathway. In vitro experiments showed that kaempferide significantly increased the expression of antioxidant genes and promoted osteogenic differentiation. Furthermore, kaempferide also improved the osteogenic differentiation inhibited by H2O2 through the enhancement of antioxidant capacity. Notably, kaempferide promoted cell antioxidant capacity by the increased nuclear translocation of FoxO1 and ß-catenin. CONCLUSIONS: These findings suggest that kaempferide is the natural antioxidant to promote osteogenesis effectively through the FoxO1/ß-catenin signaling pathway. Natural antioxidant therapy maybe a promising strategy for the prevention and treatment of OP.

Development and Validation of KASP Assays for the Genotyping of Racing Performance-Associated Single Nucleotide Polymorphisms in Pigeons.

Pigeon racing's recent upturn in popularity can be attributed in part to the huge prize money involved in these competitions. As such, methods to select pigeons with desirable genetic characteristics for racing or for selective breeding have also been gaining more interest. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping-specific genes is one of the most commonly used molecular techniques, which can be costly, laborious and time consuming. The present study reports the development of an alternative genotyping method that employs Kompetitive Allele Specific Polymerase Chain Reaction (KASP) technology with specifically designed primers to detect previously reported racing performance-associated polymorphisms within the LDHA, MTYCB, and DRD4 genes. To validate, KASP assays and PCR-RFLP assays results from 107 samples genotyped for each of the genes were compared and the results showed perfect (100%) agreement of both methods. The developed KASP assays present an alternative rapid, reliable, and cost-effective method to identify polymorphisms in pigeons.

Risk factors for inadvertent intraoperative hypothermia in patients undergoing laparoscopic surgery: A prospective cohort study.

BACKGROUND: Inadvertent intraoperative hypothermia is frequent during open surgeries; however, few studies on hypothermia during laparoscopic abdominal surgery have been reported. We aimed to investigate the incidence and risk factors for hypothermia in patients undergoing laparoscopic abdominal surgery. METHODS: This single-center prospective cohort observational study involved patients undergoing laparoscopic surgery between October 2018 and June 2019. Data on core body temperature and potential variables were collected. A multivariate logistic regression analysis was performed to identify the risk factors associated with hypothermia. A Cox regression analysis was used to verify the sensitivity of the results. RESULTS: In total, 690 patients were included in the analysis, of whom 200 (29.0%, 95% CI: 26%-32%) had a core temperature < 36°C. The core temperature decreased over time, and the incident hypothermia increased gradually. In the multivariate logistic regression analysis, age (OR = 1.017, 95% CI: 1.000-1.034, P = 0.050), BMI (OR = 0.938, 95% CI: 0.880-1.000; P = 0.049), baseline body temperature (OR = 0.025, 95% CI: 0.010-0.060; P < 0.001), volume of irrigation fluids (OR = 1.001, 95% CI: 1.000-1.001, P = 0.001), volume of urine (OR = 1.001, 95% CI: 1.000-1.003, P = 0.070), and duration of surgery (OR = 1.010, 95% CI: 1.006-1.015, P < 0.001) were significantly associated with hypothermia. In the Cox analysis, variables in the final model were age, BMI, baseline body temperature, volume of irrigation fluids, blood loss, and duration of surgery. CONCLUSIONS: Inadvertent intraoperative hypothermia is evident in patients undergoing laparoscopic surgeries. Age, BMI, baseline body temperature, volume of irrigation fluids, and duration of surgery are significantly associated with intraoperative hypothermia.

Imaging features of microwave-ablated lesions of the VX2 tumors in rabbit lungs on computed tomography and magnetic resonance images and their correlations with pathological examination.

PURPOSE: This study aimed to explore the accuracy of different imaging methods for lesion volume estimation pre- and post-microwave ablation (MWA) as compared with that of pathological examination. METHODS: We used the VX2 cell line to establish the VX2 lung tumor model in rabbits, followed by MWA of the tumor. The imaging features of the VX2 tumors were documented. The volume of the tumors and the ablated lesions were measured and compared across imaging methods, using the pathological examination as reference. RESULTS: Tumors were successfully developed in 11 rabbits (age, 13.91 ± 1.38 weeks; weight, 2.15 ± 0.56 kg). The mean volume of the tumors was 2.05 ± 1.88 cm3. CT showed the strongest correlation with the pathologic examination results (r = 0.998, p<.001). MWA created three-layered structures that were delineated on MRI. The mean volume of the post-ablation lesion was 10.39 ± 8.93 cm3, and the measurement of the post-ablation volume on 3D-VIBE-T1WI showed the strongest correlation with the pathologic examination results (r = 0.991, p<.001). CONCLUSION: Both CT and MRI are capable of depicting lung tumors. In terms of post-ablation evaluation, MR images could provide more versatile information. The 3D-VIBE-T1WI sequence provides more precise lesion volume evaluation after ablation compared with other methods.

Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV.

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.