Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has caused widespread outbreaks in poultry and has exhibited an expanding host range among avian species, accompanied by ongoing genetic variation and adaptation. As many mosquito-borne flaviviruses can infect mammals and cross species barriers, it remains unclear whether recently emerged DTMUV variants have acquired similar potential. To address this, we assessed whether the circulating variant AQ-19 can establish infection in mammals via peripheral routes. Despite its enhanced virulence in avian species, this variant failed to establish infection in mice following peripheral inoculation, indicating that a substantial barrier still restricts cross-species transmission to mammals. We next investigated whether additional adaptive changes are required for DTMUV to overcome this barrier. Through serial intracranial passage in Institute of Cancer Research (ICR) mice, we generated a mouse-adapted strain (F13) that exhibited markedly enhanced virulence and, notably, acquired the ability to invade the central nervous system (CNS) via intranasal (i.n.) inoculation, resulting in lethal infection. This neuroinvasive phenotype was strictly route-dependent, as infection remained ineffective via intraperitoneal (i.p.) and subcutaneous (s.c.) routes. We found that the adapted virus invades the CNS via the olfactory route and disseminates broadly, exhibiting strong neurotropism and inducing severe neural damage. Functional disruption of the olfactory epithelium using ZnSO4 significantly delayed disease onset and reduced viral burden in the brain, supporting its role as a major route for nasal-to-brain transmission. Importantly, genomic analysis identified only four nonsynonymous amino acid substitutions in the adapted strain, suggesting that a limited number of mutations may be sufficient to confer enhanced neurovirulence and enable neuroinvasion in a mammalian host. Collectively, these findings demonstrate that the emerging DTMUV variant AQ-19 remains restricted in mammals, whereas limited adaptive changes acquired during experimental mouse adaptation are associated with the acquisition of neuroinvasive capacity, providing new insights into the evolutionary potential of DTMUV for mammalian adaptation.

Virus-like particles (VLPs) are engineered nanoplatforms that mimic viral structures, offering high immunogenicity, biocompatibility, and functional versatility for cancer immunotherapy. While widely explored in human oncology as nanovaccines and targeted delivery systems for chemo-/immuno-therapeutics and genetic payloads (e.g., mRNA, siRNA, and CRISPR/Cas systems), their potential in veterinary oncology remains underexploited. This review synthesizes recent advances in VLP design, including scaffold engineering, antigen display, cargo encapsulation, and surface functionalization, and discusses the mechanistic basis of VLP-induced antitumor immunity, encompassing dendritic cell activation, adaptive immune amplification, and tumor microenvironment remodeling. Importantly, we highlight the emerging role of companion animals with spontaneous tumors-such as lymphoma, melanoma, and mammary carcinoma-as immunocompetent translational models within the One Health framework. Comparative oncology reveals striking parallels in oncogenic pathways, immune landscapes, and therapeutic responses, supporting the use of canine and feline cancers as biologically relevant intermediates between murine studies and human clinical trials. We provide an evidence-based assessment of representative VLP platforms, evaluate their translational readiness, and examine cross-species opportunities for shared target development, biomarker discovery, and regulatory convergence, while also addressing species-specific biological and technical limitations. Finally, we propose a forward-looking roadmap that prioritizes manufacturing standardization, biomarker development, comparative validation, precision engineering, and emerging technologies such as AI-guided design and tumor-on-chip systems. Collectively, we position One Health as an operational strategy to accelerate the bidirectional translation of VLP-based immunotherapies for both human and veterinary cancer patients.

Canine distemper, a fatal and highly transmissible disease caused by the canine distemper virus (CDV), poses a major threat to the companion animal industry. An urgent need exists for a rapid, specific, and simple method for the detection of this disease in order to improve its prevention and control. In this research, two monoclonal antibodies (mAbs), 1D3E9 and 1H9B7, were prepared, both of which specifically recognize the nucleoprotein (N protein) of CDV, and an immunochromatographic assay for CDV detection was subsequently developed using these mAbs. The results showed that both mAbs belong to the IgG1 subclass with kappa light chains. 1D3E9 was found to recognize the linear epitope 410AGPKQSQITFLH421, while 1H9B7 targeted the epitope 450HFNDERFPGH459. The test strips exhibited high specificity and good stability for up to two months when stored at 4, 25, and 37 °C. The assay exhibited a sensitivity of 102.39 TCID50/0.1 mL. When compared with RT-PCR for detecting CDV in clinical samples, the concordance rate was 91.67%. Thus, this method shows great potential for facilitating rapid on-site detection of CDV and could be highly beneficial from the viewpoint of disease surveillance and control.

Mycoplasma synoviae (MS) is considered to be one of the main mycoplasma pathogens of poultry, causing arthritis, airsacculitis, eggshell apex abnormalities and production drops in chickens and turkeys. Infection by MS usually results in considerable economic losses to the poultry industry worldwide. Therefore, it is essential to develop a highly sensitive and accurate diagnostic method in the livestock production. The MSLP53 was predicted as a highly conserved and specific membrane associated lipoprotein of MS by bioinformatics analysis. The His-tagged MSLP53 (rMSLP53) protein was expressed and purified using E. coli expression system, and was confirmed by Western blotting to react with each MS-positive serum, but not react with positive sera against other avian pathogens, suggesting that the rMSLP53 had strong immunoreactivity and specificity. An rMSLP53-based indirect ELISA was developed, compared to IDEXX kit with a pool of 277 chicken sera samples, and showed high sensitivity (85.54

Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.

Felis domesticus allergen 1 (Fel d 1), the primary allergen produced by cats, is a key trigger of various allergic symptoms in humans, such as rhinitis, conjunctivitis, and asthma. In this study, a recombinant Fel d 1 protein composed of chain 2 and chain 1 was successfully produced in E. coli, based on a previously reported fusion construct that closely approximates the native allergen. Two antibodies, 2A7 and 6B1, were selected for further study due to their high specificity and complementary epitope recognition. A dual-antibody sandwich ELISA was established using 2A7 as the capture antibody and HRP-conjugated 6B1 as the detection antibody. The assay showed a detection range of 10-500 ng/mL with a sensitivity of 1 ng/mL. Application to feline saliva and fur samples revealed detectable Fel d 1 levels, and results correlated well with commercial kits. Epitope mapping identified distinct α-helical regions targeted by 2A7 and 6B1, and sequence analysis of their variable regions confirmed diverse V(D)J gene usage. This study provides a valuable antibody pair and a sensitive ELISA method for Fel d 1 quantification, with potential applications in allergen exposure assessment and diagnostic development. SCIENTIFIC SECTION: Immunology, Antibodies, and Diagnostics.

Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.

Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor. In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 102 copies/µL. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 100 copies/µL. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 102 copies/µL. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results. We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.

Duck Tembusu virus (DTMUV) is a significant etiological agent responsible for egg production decline in laying ducks and retarded growth in ducklings, contributing to considerable economic losses in the poultry sector. Current research mainly focuses on the development of vaccines for the prevention of DTMUV. However, there is relatively little research on antiviral drugs against this virus. Curcumin has been reported to exert antiviral effects against multiple viruses. However, its efficacy against DTMUV remains unclear. This study aimed to investigate the antiviral activity of curcumin against DTMUV both in vitro and in vivo. Cytotoxicity in BHK-21 cells was assessed utilizing the Cell Counting Kit-8 (CCK-8) assay. The antiviral effects of curcumin were evaluated using viral titration, qRT-PCR, Western blotting, and indirect immunofluorescence. In addition, a DTMUV-infected duck model was used to assess the therapeutic potential of curcumin in vivo. The experimental results indicated that curcumin was non-toxic to BHK-21 cells at concentrations up to 30 μM. In vitro, curcumin significantly reduced DTMUV replication in a dose-dependent manner, as confirmed by decreased viral titers, RNA copy numbers, and E protein expression. Further analysis revealed that curcumin exerted its antiviral effect primarily at the post-adsorption stage of the viral life cycle. In vivo, curcumin treatment improved the survival rate of DTMUV-infected ducklings and alleviated virus-associated pathology. These findings demonstrate that curcumin effectively inhibits DTMUV infection and highlight its potential as a viable candidate for the prevention and treatment of DTMUV-related diseases.

A novel goose astrovirus (GAstV) has broken out across China in recent years, causing widespread damage to the poultry industry. In goslings infected with GAstV, the leading cause of death is visceral gout. However, our understanding of the mechanism of gout formation in GAstV infection is largely inadequate. The aim of this study was to examine the pathogenicity of a GAstV strain and explore the molecular mechanisms of visceral gout caused by viral infection in goslings. The virulent GAstV strain HR2105/1 was effectively isolated from the visceral tissue of goslings in gout-affected areas. The whole genome of the HR2105/1 strain was sequenced and analyzed. Subsequently, we established a gosling gout models with experimental GAstV infection. Finally, we conducted a study on the mechanism of GAstV induced acute kidney injury. Phylogenetic analysis of the complete genome sequence showed that it was closely related to the strain circulating in China since 2016, and it was grouped within the GAstV-1 cluster. The clinical signs were reproduced by experimental infection of healthy goslings with the isolated strain and were found to be similar to those reported in clinical cases. Moreover, the virus exhibits strong renal tropism. Infection with the GAstV strain HR2105/1 was found to cause acute kidney injury, as evidenced by increased levels of uric acid and creatinine as well as severe pathological damage. Mechanistic experiments with Masson and Picrosirius Red staining revealed fibrosis in renal tissues after GAstV infection. Furthermore, TUNEL staining revealed that GAstV infection triggered renal cell apoptosis. Additionally, RT-qPCR revealed that GAstV infection caused an excessive inflammatory response by upregulating the expression of IL-1β, IL-6, IL-10, TGF-β, and iNOS in renal tissues. Overall, our findings demonstrate that GAstV infection causes renal damage by inducing renal cell apoptosis, fibrosis, and excessive inflammatory response, which subsequently leads to hyperuricemia and lethal visceral gout formation. This is the first systematic study on the etiology of lethal gout in goslings caused by GAstV infection, and we believe that the findings can guide vaccine development and therapeutic targets for GAstV-associated renal diseases.

Ferroptosis is a form of regulated cell death characterized by massive iron accumulation and iron-dependent lipid peroxidation, differing from apoptosis, necroptosis, and autophagy in several aspects. Ferroptosis is regarded as a critical mechanism of a series of pathophysiological reactions after stroke because of iron overload caused by hemoglobin degradation and iron metabolism imbalance. In this review, we discuss ferroptosis-related metabolisms, important molecules directly or indirectly targeting iron metabolism and lipid peroxidation, and transcriptional regulation of ferroptosis, revealing the role of ferroptosis in the progression of stroke. We present updated progress in the intervention of ferroptosis as therapeutic strategies for stroke in vivo and in vitro and summarize the effects of ferroptosis inhibitors on stroke. Our review facilitates further understanding of ferroptosis pathogenesis in stroke, proposes new targets for the treatment of stroke, and suggests that more efforts should be made to investigate the mechanism of ferroptosis in stroke.

Duck Tembusu virus (DTMUV) is a newly emerging pathogen that causes massive economic losses to the poultry industry in China and neighbouring countries. Vimentin, an intermediate filament protein, has been demonstrated to be involved in viral replication during infection. However, the specific role of vimentin in DTMUV replication has not been determined. In this study, we found that overexpression of vimentin in BHK-21 cells can inhibit DTMUV replication. Moreover, DTMUV replication was enhanced after vimentin expression was reduced in BHK-21 cells via small interfering RNA (siRNA). Further research indicated that DTMUV infection had no effect on the transcription or expression of vimentin. However, we found that DTMUV infection induced vimentin rearrangement, and the rearrangement of vimentin was subsequently confirmed to negatively modulate viral replication through the use of a vimentin network disrupting agent. Vimentin rearrangement is closely associated with its phosphorylation. Our experiments revealed that the phosphorylation of vimentin at Ser56 was promoted in the early stage of DTMUV infection. In addition, by inhibiting the phosphorylation of vimentin at Ser56 with a CDK5 inhibitor, vimentin rearrangement was suppressed, and DTMUV replication was significantly enhanced. These results indicated that DTMUV infection induced vimentin phosphorylation and rearrangement through CDK5, resulting in the inhibition of DTMUV replication. In summary, our study reveals a role for vimentin as a negative factor in the process of DTMUV replication, which helps to elucidate the function of cellular proteins in regulating DTMUV replication.

Abstract Duck Tembusu virus (DTMUV) is a neurotropic virus belonging to the genus Flavivirus , and causes massive economic losses to the poultry industry in China and neighbouring countries. Autophagy plays a pivotal role in cellular responses to pathogens and viral pathogenesis. However, little is known about the roles of autophagy in DTMUV replication and virus pathogenesis, especially in neuropathogenesis. In this study, mouse neuroblastoma cells (Neuro-2a) were used to establish a DTMUV infection cell model. Our experiments indicated that DTMUV infection induced incomplete autophagy in Neuro-2a cells. Then, autophagy induced by DTMUV was altered with different autophagy regulators, and we found that incomplete autophagy promoted DTMUV replication. Furthermore, we showed that DTMUV infection activated both the ERK and AMPK pathways, resulting in decreased phosphorylation levels of the autophagy repressor mTOR, subsequently leading to autophagic induction. In addition, we utilized ICR mice as the DTMUV-infected animal model to evaluate the autophagic responses in brain tissues and investigate the effects of autophagy on viral replication and tissue lesions. As a result, we confirmed that DTMUV induced incomplete autophagy in mouse brain tissues and that autophagy inducer treatment promoted DTMUV replication and aggravated DTMUV-induced lesions, whereas autophagy inhibitor treatment led to the opposite effects. Collectively, DTMUV infection induced incomplete autophagy through the ERK/mTOR and AMPK/mTOR signalling pathways to promote viral replication in mouse neuronal cells, and DTMUV-induced incomplete autophagy contributed to the neuropathogenesis of DTMUV.

Variations in the pathogenicity of Newcastle disease virus (NDV), the agent causing Newcastle disease, are associated with variants of different virulence. A few studies have characterized the expression of microRNAs (miRNAs) in NDV-infected avian cells. Here, the expression of miRNAs in chicken embryo fibroblasts (CEFs) infected with Herts/33 and LaSota NDV strains (highly virulent and nonvirulent, respectively) was determined using RNA sequencing. miRNAs involved in NDV infection included 562 previously documented and 184 novel miRNAs. miRNA target genes involved transcription factors, cell apoptosis, ubiquitin-mediated proteolysis, and protein processing in the endoplasmic reticulum. Potential target genes associated with autophagy were verified by qRT-PCR. No studies have documented the miRNA profiles of CEFs infected with NDVs variants. This study adds to our knowledge of the cellular miRNAs involved in NDV infection and the complex molecular mechanisms mediating virus-host interactions. The results of this study will aid the development of strategies against the chicken virus.

Duck Tembusu virus (DTMUV) is a neurotropic virus in the genus Flavivirus that causes massive economic losses to the poultry industry in China and neighbouring countries. Autophagy is pivotal in cellular responses to pathogens and in viral pathogenesis. However, little is known about the roles of autophagy in DTMUV replication and viral pathogenesis, especially in neuropathogenesis. In this study, mouse neuroblastoma cells (Neuro-2a) were used to establish a cell model of DTMUV infection. Our experiments indicated that DTMUV infection induced incomplete autophagy in Neuro-2a cells. Then, we used different autophagy regulators to alter the autophagy induced by DTMUV and found that incomplete autophagy promoted DTMUV replication. Furthermore, we showed that DTMUV infection activated the ERK and AMPK pathways, resulting in decreased phosphorylation of the autophagy repressor mTOR, subsequently leading to autophagic induction. In addition, we utilized ICR mice in an animal model of DTMUV infection to evaluate the autophagic responses in brain tissues and investigate the effects of autophagy on viral replication and tissue lesions. Our results confirmed that DTMUV induced incomplete autophagy in mouse brain tissues and that autophagy inducer treatment promoted DTMUV replication and aggravated DTMUV-induced lesions, whereas autophagy inhibitor treatment had the opposite effects. In summary, DTMUV infection induced incomplete autophagy through the ERK/mTOR and AMPK/mTOR signalling pathways to promote viral replication in mouse neuronal cells, and DTMUV-induced incomplete autophagy contributed to the neuropathogenesis of DTMUV.

To reveal the etiological and genetic characteristics of the novel goose astrovirus epidemic strains causing goose gout in recent years,the samples were collected from goslings suspected to be infected with the novel goose astrovirus in Anhui Province,China.The viruses were isolated by using healthy goose embryos,and the whole genome sequences of the isolated viruses were determined and analyzed.The results showed that five samples infected with novel goose astrovirus were identified.Five strains of goose astrovirus were successfully isolated from goose embryos by blind transmission for 3 generations.The strains were named as AHAU2018,DY-19,ZY02,HR2105/2 and HR2100/1,respectively.The whole genome sequencing results showed that the total genome length of the isolated strains was 7 175 nt.Phylogenetic tree analysis showed that the five isolated strains belonged to the novel goose astrovirus causing goose gout,and the novel goose astrovirus isolated before 2019 and after 2019 were in different evolutionary sub-branches.The novel goose astrovirus has been mutated in China,and new evolutionary sub branches have emerged.But,whether the dominant genotype has changed remains to be further studied.

Since 2010, the Tembusu virus (TMUV) has been highly prevalent in China, causing significant economic losses to the poultry industry. In 2022, a suspected outbreak of TMUV occurred at a goose farm located in Anhui Province. A strain of TMUV, TMUV HQ-22, was isolated from the infected geese. Phylogenetic analysis using the E gene of the HQ-22 strain demonstrated its affiliation with cluster 3, a less commonly reported cluster in comparison to the main circulating cluster, cluster 2. Through a comparison of the envelope (E) protein of HQ-22 with other typical TMUV strains, a mutation at the 157th amino acid position was identified, wherein valine (V) in cluster 3 changed to alanine (A), a characteristic that is unique to cluster 2. These findings highlight the diversity and complexity of the TMUV strains circulating in China. In our experimental analysis, an injection of TMUV HQ-22 into the muscles of 3-day-old goslings resulted in severe neurological symptoms and a mortality rate of 60%. Similarly, the intracranial or intranasal infection of 3-week-old ICR mice with TMUV HQ-22 led to severe neurological symptoms and respective mortality rates of 100% or 10%. In summary, our study isolated a TMUV strain, TMUV HQ-22, from geese that belongs to cluster 3 and exhibits significant pathogenicity in both goslings and ICR mice. These results emphasize the genetic diversity of the TMUV circulating in China and expand the host range beyond mosquitoes to include ducks, chickens, geese, and even mice. It is crucial to not underestimate the risk of TMUV infection in mammals, warranting our utmost attention.

BACKGROUND:Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX's potential as a diagnostic antigen and its role in MS cytoadherence.RESULTS:Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin.CONCLUSION:MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) to prepare polyclonal antibody and identify specificity. Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection. Viperin gene, cloned from mouse brain tissue by reverse transcription PCR, was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E. coli Rosetta. The recombinant viperin protein was induced by isopropyl thiogalactoside (IPTG) and its solubility was analyzed. The protein was purified by potassium chloride (KCl) staining and gel cutting method. Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen, and the titer of polyclonal antibody was determined by indirect ELISA. Western blot analysis and indirect fluorescence assay (IFA) were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein. Results The mouse viperin gene was successfully cloned and the viperin protein was expressed. The titer of the prepared anti-viperin polyclonal antibody reached 1:25 600. The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells. Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.

Duck tembusu virus (DTMUV), which causes huge economic losses for the poultry industries in Southeast Asia and China, was first identified in 2010. DTMUV disease has become an important disease that endangers the duck industry. A sensitive, accurate, and convenient DTMUV detection method is an important means to reduce the occurrence of the disease. In this study, a CRISPR/Cas13a system was combined with recombinase polymerase amplification to develop a convenient diagnostic method to detect DTMUV. The novel method was based on isothermal detection at 37°C, and the detection was used for visual readout or real-time analysis. The assay was highly sensitive and specific, with a detection limit of 1 copy/μL of the target gene and showed no cross-reactivity with other pathogens. The enhanced Cas13a detection worked well with clinical samples. Overall, a visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a proved to be a powerful tool for detecting DTMUV.