YAP in development and disease: Navigating the regulatory landscape from retina to brain.

The distinctive role of Yes-associated protein (YAP) in the nervous system has attracted widespread attention. This comprehensive review strategically uses the retina as a vantage point, embarking on an extensive exploration of YAP's multifaceted impact from the retina to the brain in development and pathology. Initially, we explore the crucial roles of YAP in embryonic and cerebral development. Our focus then shifts to retinal development, examining in detail YAP's regulatory influence on the development of retinal pigment epithelium (RPE) and retinal progenitor cells (RPCs), and its significant effects on the hierarchical structure and functionality of the retina. We also investigate the essential contributions of YAP in maintaining retinal homeostasis, highlighting its precise regulation of retinal cell proliferation and survival. In terms of retinal-related diseases, we explore the epigenetic connections and pathophysiological regulation of YAP in diabetic retinopathy (DR), glaucoma, and proliferative vitreoretinopathy (PVR). Lastly, we broaden our exploration from the retina to the brain, emphasizing the research paradigm of "retina: a window to the brain." Special focus is given to the emerging studies on YAP in brain disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), underlining its potential therapeutic value in neurodegenerative disorders and neuroinflammation.

Deciphering the potential role of PGRN in regulating CD8+ T cell antitumor immunity.

A key factor contributing to resistance in immune checkpoint blockade (ICB) therapies is CD8+ T-cell tolerance in the tumor microenvironment (TME), partly resulting from upregulating coinhibitory receptors. Here, we describe the role of PGRN as a coinhibitory molecule that modulates the antitumor response of CD8+ T cells, thus presenting a novel immunosuppressive target for lung cancer. The in vivo subcutaneous transplanted lung cancer model showed that PGRN expression was elevated on CD8+ T cells that infiltrated transplanted lung cancers. Furthermore, PGRN deficiency was found to specifically encourage the infiltration of CD8+ T cells, enhance their proliferation, migration, and activation, and resist apoptosis, ultimately inhibiting tumor growth. This was achieved by PGRN knockout, increasing the production of T cell chemokine CCL3, which boosts the antitumor immune response induced by CD8+ T cells. Critically, the PD-L1 inhibitor exhibited a synergistic effect in enhancing the antitumor response in PGRN-/- mice. In summary, our findings highlight the significance of PGRN as a novel target for boosting CD8+ T cells antitumor immunity and its potential to overcome the resistance in ICB therapy.

The angelica Polysaccharide: a review of phytochemistry, pharmacology and beneficial effects on systemic diseases.

Angelica sinensis is a perennial herb widely distributed around the world, and angelica polysaccharide (APS) is a polysaccharide extracted from Angelica sinensis. APS is one of the main active components of Angelica sinensis. A large number of studies have shown that APS has hematopoietic, promoting blood circulation, radiation resistance, lowering blood glucose, enhancing the body immunity and other pharmacological effects in a variety of diseases. However, different extraction methods and extraction sites greatly affect the efficacy of APS. In recent years, with the emerging of new technologies, there are more and more studies on the combined application and structural modification of APS. In order to promote the comprehensive development and in-depth application of APS, this narrative review systematically summarizes the effects of different drying methods and extraction sites on the biological activity of APS, and the application of APS in the treatment of diseases, hoping to provide a scientific basis for the experimental study and clinical application of APS.

Opportunities and perspectives of small molecular phosphodiesterase inhibitors in neurodegenerative diseases.

Phosphodiesterase (PDE) is a superfamily of enzymes that are responsible for the hydrolysis of two second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). PDE inhibition promotes the gene transcription by activating cAMP-response element binding protein (CREB), initiating gene transcription of brain-derived neurotrophic factor (BDNF). The procedure exerts neuroprotective profile, and motor and cognitive improving efficacy. From this point of view, PDE inhibition will provide a promising therapeutic strategy for treating neurodegenerative disorders. Herein, we summarized the PDE inhibitors that have entered the clinical trials or been discovered in recent five years. Well-designed clinical or preclinical investigations have confirmed the effectiveness of PDE inhibitors, such as decreasing Aß oligomerization and tau phosphorylation, alleviating neuro-inflammation and oxidative stress, modulating neuronal plasticity and improving long-term cognitive impairment.

Anthocyanin biosynthesis in goji berry is inactivated by deletion in a bHLH transcription factor LrLAN1b promoter.

Black goji berry (Lycium ruthenicum Murray) contains a rich source of health-promoting anthocyanins which are used in herbal medicine and nutraceutical foods in China. A natural variant producing white berries allowed us to identify two key genes involved in the regulation of anthocyanin biosynthesis in goji berries: one encoding a MYB transcription factor (LrAN2-like) and one encoding a basic helix-loop-helix (bHLH) transcription factor (LrAN1b). We previously found that LrAN1b expression was lost in the white berry variant, but the molecular basis for this phenotype was unknown. Here, we identified the molecular mechanism for loss of anthocyanins in white goji berries. In white goji, the LrAN1b promoter region has a 229-bp deletion that removes 3 MYB-binding elements and 1 bHLH-binding element, which are key to its expression. Complementation of the white goji berry LrAN1b allele with the LrAN1b promoter restored pigmentation. Virus-induced gene silencing of LrAN1b in black goji berry reduced fruit anthocyanin biosynthesis. Molecular analyses showed that LrAN2-like and another bHLH transcription factor LrJAF13 can activate LrAN1b by binding directly to the MYB-recognizing element (MRE) and bHLH-recognizing element (BRE) of its promoter-deletion region. LrAN1b expression is enhanced by the interaction of LrAN2-like with LrJAF13 and the WD40 protein LrAN11. LrAN2-like and LrAN11 interact with either LrJAF13 or LrAN1b to form two MYB-bHLH-WD40 (MBW) complexes, which hierarchically regulate anthocyanin biosynthesis in black goji berry. This study on a natural variant builds a comprehensive anthocyanin regulatory network that may be manipulated to tailor goji berry traits.

The repeated administration of rhIL-12 for 14 weeks in rhesus monkeys: A toxicity assessment.

Interleukin-12 (IL-12) is known to exert antitumor immune effects by promoting the activation and proliferation of T cells and NK cells within the immune system. However, clinical trials have observed systemic toxicity associated with the administration of IL-12. This has shelved development plans for its use as a cancer therapeutic drug. Therefore, it is critical that we perform a systematic evaluation of the toxicity and safety of repeated IL-12 administration. In this study, we conducted a comprehensive evaluation of the toxicity and safety of repeated rhIL-12 (recombinant human interleukin-12) administration in rhesus monkeys by assessing its effects on the immune system, organ function, and vital signs. Rhesus monkeys were subcutaneously injected with 0.5, 2.5, and 12.5 µg/kg of rhIL-12 for up to for 14 consecutive weeks. The low dose exhibited no signs of toxicity, whereas animals receiving higher doses displayed symptoms such as loose stools, reduced activity, anemia, and elevated liver function indicators (AST and TBIL). Following three administrations of 12.5 µg/kg, high dosing was adjusted to 7.5 µg/kg due to manifestations of symptoms like loose stools, decreased activity, and huddling in the cage. Furthermore, rhesus monkeys exhibited marked immunogenic responses to recombinant human interleukin-12 (rhIL-12). However, based on overall study findings, the No Observed Adverse Effect Level (NOAEL) for the subcutaneous injection of rhIL-12, when repeatedly administered for 3 months in rhesus monkeys, was considered to be 0.5 µg/kg. The Highest Non-Severely Toxic Dose (HNSTD) was considered to be 7.5 µg/kg.

In Vivo Microwave-Induced Thermoacoustic Endoscopy for Colorectal Tumor Detection in Deep Tissue.

Optical endoscopy, as one of the common clinical diagnostic modalities, provides irreplaceable advantages in the diagnosis and treatment of internal organs. However, the approach is limited to the characterization of superficial tissues due to the strong optical scattering properties of tissue. In this work, a microwave-induced thermoacoustic (TA) endoscope (MTAE) was developed and evaluated. The MTAE system integrated a homemade monopole sleeve antenna (diameter = 7 mm) for providing homogenized pulsed microwave irradiation to induce a TA signal in the colorectal cavity and a side-viewing focus ultrasonic transducer (diameter = 3 mm) for detecting the TA signal in the ultrasonic spectrum to construct the image. Our MTAE, system combined microwave excitation and acoustic detection; produced images with dielectric contrast and high spatial resolution at several centimeters deep in soft tissues, overcome the current limitations of the imaging depth of optical endoscopy and mechanical wave-based imaging contrast of ultrasound endoscopy, and had the ability to extract complete features for deep location tumors that could be infiltrating and invading adjacent structures. The practical feasibility of the MTAE system was evaluated i n vivo with rabbits having colorectal tumors. The results demonstrated that colorectal tumor progression could be visualized from the changes in electromagnetic parameters of the tissue via MTAE, showing its potential clinical application.

[Acute toxicity test of the Li-Dan-He-Ji granules].

OBJECTIVE: To observe the acute toxic reaction of the Li-Dan-He-Ji granules, and to evaluate its safety. METHODS: Sixty C57BL6/J mice were randomly divided into normal control group, vehicle group and drug treatment group, with 10 females and 10 males in each group. According to the Technical guidelines for the study of toxicity of single drug administration, the maximum administration dosage (MAD) was used to intragastric administration of Li-Dan-He-Ji granules 0.04 mL/g (42.8 g/kg), three times within 24 hours, with an interval of 6 hours. The vehicle group was fed with the same pure water. The normal control group received no treatment. The mice were observed continuously for 14 days, and the appearance characteristics, behavioral activities, body weight changes and the number of deaths in each group were recorded. At the 14 days, blood samples were collected from the eyeballs, and routine blood tests such as white blood cell count (WBC), lymphocyte count (LYM), neutrophil count (NEU), lymphocyte percentage (LYM%), neutrophil percentage (NEU%), red blood cell count (RBC), hemoglobin (Hb), and platelet count (PLT) were performed. And alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr) and other biochemical indicators. The mice were then sacrificed, and the histopathological changes of liver and kidney were observed by hematoxylin-eosin (HE) staining. The organ indexes of heart, liver, spleen, lung, kidney and thymus were calculated. RESULTS: The median lethal dose (LD50) of Li-Dan-He-Ji granules were not obtained. During the MAD experiment, the animals in each group did not die, their behavioral activities were normal, and there was no significant change in liver and kidney histopathological examination. There were no significant differences in body weight, blood routine, biochemical indexes and organ index among all groups (all P > 0.05). The body weight (g) of normal control female and male group, vehicle female and male group and drug female and male group before administration were 18.96±1.14, 19.65±1.45, 19.33±1.30, 19.53±1.22, 19.28±1.69 and 19.48±1.28; 14 days after administration were 27.69±0.81, 28.19±2.22, 27.77±1.00, 27.88±1.85, 27.92±1.33 and 28.07±1.93, respectively. CONCLUSIONS: The Li-Dan-He-Ji granules have low oral toxicity, combined with clinical observation, can be safely used in infants.

Analysis of risk factors for non-alcoholic fatty liver disease in hospitalized children with obesity before the late puberty stage.

Objective: This study aimed to determine the clinical characteristics of obese pediatric non-alcoholic fatty liver disease (NAFLD) in central China and verify the applicability of some known risk factors for pediatric NAFLD before late puberty. Methods: This was a retrospective case-control study. A total of 1,029 inpatients at Wuhan Children's Hospital before the late puberty stage were enrolled in the study, including 815 children with obesity (non-NAFLD group) and 214 children with obesity and NAFLD (NAFLD group) diagnosed by liver ultrasound. Subgroup analyses were performed according to sex and puberty. The anthropometric indices and laboratory test data of these 1,029 children were sorted. After intergroup comparison, a logistic regression model was used to determine the risk factors for pediatric NAFLD. Significant risk factors for NAFLD were further tested using receiver operating characteristic (ROC) curves to evaluate their ability to predict an early diagnosis of NAFLD. Results: The NAFLD group had a mean age of 11.03 ± 1.66, with 11.18 ± 1.66 and 10.27 ± 1.45 years for male and female children, respectively (p < 0.05 and p < 0.01, respectively). Even subdivided by both sex and puberty, raised body mass index (BMI), homeostatic model-insulin resistance, triglycerides, alanine transaminase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were still found in the non-NAFLD and NAFLD groups (p < 0.05 and p < 0.01, respectively). The results of logistic regression analysis showed that BMI (odds ratio [OR], 1.468;95% confidence interval [CI], 1.356-1.590; p<0.001) and ALT (OR, 1.073;95%CI, 1.060-1.087; P<0.001) were two most independent risk factors for NAFLD. The maximal OR for BMI was 1.721 (95% CI, 1.336-2.217). In the female group, the maximal OR of ALT was found to be 1.104 (95% CI, 1.061-1.148). Age and thyroid-stimulating hormone (TSH) and γ-GT levels were also risk factors, but they appeared only in some groups. The results of the ROC analysis showed that ALT was a better predictor of pediatric NAFLD than BMI. The maximum area under the ROC curve in six of the nine groups belongs to ALT. Conclusions: BMI, ALT, and age are risk factors for NAFLD in children with obesity before late puberty. BMI had the greatest exposure risk for NAFLD, and ALT had the highest predictive value for the diagnosis of NAFLD. At the stratified level, for exposure risk, age was specific to the male sex, TSH was specific to the early puberty stage, and γ-GT was specific to the female sex plus the prepuberty stage. On a stratified level, for the female sex, even with age stratification, BMI rather than ALT has a better ability for the diagnosis of NAFLD.

A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTRN-S1NCOE to obtain vC/SM3'UTRN-tPAsS1NCOE and vC/SM3'UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-ß induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-ß induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity.

Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment.

Virtual Screening of Nrf2 Dietary-Derived Agonists and Safety by a New Deep-Learning Model and Verified In Vitro and In Vivo.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an essential regulatory target of antioxidants, but the lack of Nrf2 active site information has hindered discovery of new Nrf2 agonists from food-derived compounds by large-scale virtual screening. Two deep-learning models were separately trained to screen for Nrf2-agonists and safety. The trained models screened potentially active chemicals from approximately 70,000 dietary compounds within 5 min. Of the 169 potential Nrf2 agonists identified via deep-learning screening, 137 had not been reported before. Six compounds selected from the new Nrf2 agonists significantly increased (p < 0.05) the activity of Nrf2 on carbon tetrachloride (CCl4)-intoxicated HepG2 cells (nicotiflorin (99.44 ± 18.5%), artemetin (97.91 ± 8.22%), daidzin (87.73 ± 3.77%), linonin (74.27 ± 5.73%), sinensetin (72.74 ± 10.41%), and tectoridin (77.78 ± 4.80%)), and their safety were demonstrated by an MTT assay. The safety and Nrf2 agonistic activity of nicotiflorin, artemetin, and daidzin were also reconfirm by a single-dose acute oral toxicity study and CCl4-intoxicated rat assay.

Weakly aligned Ti3C2Tx MXene liquid crystals: measuring residual dipolar coupling in multiple co-solvent systems.

Residual Dipolar Coupling (RDC), acquired relying on weakly alignment media, is highly valuable for the structural elucidation of organic molecules. Arising from the striking features of no background signals and low critical concentrations, two-dimensional (2D) liquid crystals (LCs) show the clear advantages of acting as alignment media to measure RDCs. So far, creating multisolvent compatible 2D LC media through a simple and versatile method is still formidably challenging. Herein, we report the rapid creation of aligned media based on the Ti3C2Tx MXene, which self-aligned in multiple co-solvents including CH3OH-H2O, DMSO-H2O, DMF-H2O, and acetone-H2O. We demonstrated the applicability of these aligned media for the RDC measurement of small organic molecules with different polarities and solubilities. Notably, Ti3C2Tx MXene LCs without chemical modification enabled RDC measurements on aromatic molecules. The straightforward preparation of Ti3C2Tx media and its compatibility with multiple solvents will push RDC measurement as a routine methodology for structural elucidation. It may also facilitate the investigation of solvation effects on conformational dynamics.

Antitumor effect of neoantigen-reactive T cells combined with PD1 inhibitor therapy in mouse lung cancer.

PURPOSE: Neoantigens produced from mutations in tumors are important targets of T-cell-based immunotherapy and immune checkpoint blockade has been approved for treating multiple solid tumors. We investigated the potential benefit of adoptive neoantigen-reactive T (NRT) cells in combination with programmed cell death protein 1 inhibitor (anti-PD1) for treating lung cancer in a mouse model. METHODS: NRT cells were prepared by co-culturing T cells and neoantigen-RNA vaccine-induced dendritic cells. Then, adoptive NRT cells in combination with anti-PD1 were administered to tumor-bearing mice. Pre- and post-therapy cytokine secretion, antitumor efficacy, and tumor microenvironment (TME) changes were determined both in vitro and in vivo. RESULTS: We successfully generated NRT cells based on the 5 neoantigen epitopes identified in this study. NRT cells exhibited an enhanced cytotoxic phenotype in vitro and the combination therapy led to attenuated tumor growth. In addition, this combination strategy downregulated the expression of the inhibitory marker PD-1 on tumor-infiltrating T cells and promoted the trafficking of tumor-specific T cells to the tumor sites. CONCLUSION: The adoptive transfer of NRT cells in association with anti-PD1 therapy can exert an antitumor effect on lung cancer, and is a feasible, effective, and novel immunotherapy regimen for treating solid tumors.

RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) serve as critical mediators of cell-to-cell communication. Manzamine A (MA), a unique marine-derived alkaloid with various bioactivities, exerts anticancer effects against several kinds of tumors, but it remains unclear whether it has the same activity against breast cancer. Here, we proved that MA inhibits MDA-MB-231 and MCF-7 cell proliferation, migration, and invasion in a time- and dose-dependent manner. In addition, MA promotes autophagosome formation but suppresses autophagosome degradation in breast cancer cells. Importantly, we also found that MA stimulates sEVs secretion and increases autophagy-related protein accumulation in secreted sEVs, further potentiated by autophagy inhibitor chloroquine (CQ). Mechanistically, MA decreases the expression level of RIP1, the key upstream regulator of the autophagic pathway, and reduces the acidity of lysosome. Overexpression of RIP1 activated AKT/mTOR signaling, thus attenuating MA-induced autophagy and the corresponding secretion of autophagy-associated sEVs. Collectively, these data suggested that MA is a potential inhibitor of autophagy by preventing autophagosome turnover, and RIP1 mediates MA-induced secretory autophagy, which may be efficacious for breast cancer treatment.

Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6ß. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

Microglia: The breakthrough to treat neovascularization and repair blood-retinal barrier in retinopathy.

Microglia are the primary resident retinal macrophages that monitor neuronal activity in real-time and facilitate angiogenesis during retinal development. In certain retinal diseases, the activated microglia promote retinal angiogenesis in hypoxia stress through neurovascular coupling and guide neovascularization to avascular areas (e.g., the outer nuclear layer and macula lutea). Furthermore, continuously activated microglia secrete inflammatory factors and expedite the loss of the blood-retinal barrier which causes irreversible damage to the secondary death of neurons. In this review, we support microglia can be a potential cellular therapeutic target in retinopathy. We briefly describe the relevance of microglia to the retinal vasculature and blood-retinal barrier. Then we discuss the signaling pathway related to how microglia move to their destinations and regulate vascular regeneration. We summarize the properties of microglia in different retinal disease models and propose that reducing the number of pro-inflammatory microglial death and conversing microglial phenotypes from pro-inflammatory to anti-inflammatory are feasible for treating retinal neovascularization and the damaged blood-retinal barrier (BRB). Finally, we suppose that the unique properties of microglia may aid in the vascularization of retinal organoids.

Full-Color Emissive D-D-A Carbazole Luminophores: Red-to-NIR Mechano-fluorochromism, Aggregation-Induced Near-Infrared Emission, and Application in Photodynamic Therapy.

Invited for the cover of this issue are Zhao Chen at Jiangxi Science and Technology Normal University and Yue Sun at Tiangong University. For this cover picture, yellow ball containing character "D" represents donor, green ball containing character "A" represents acceptor. The image depicts eight donor-donor-acceptor-type carbazole derivatives that display full-color emission, and a luminogen that shows red to near-infrared mechano-fluorochromism and a photodynamic therapy effect. Read the full text of the article at 10.1002/chem.202203797.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.