Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Is Lethal to Neonatal Mice.

Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. We rescued synthetic wild-type SADS-CoV using one-step assembly of a viral cDNA clone by homologous recombination in yeast. Furthermore, we characterized SADS-CoV replication in vitro and in neonatal mice. We found that SADS-CoV caused severe watery diarrhea, weight loss, and a 100% fatality rate in 7- and 14-day-old mice after intracerebral infection. We also detected SADS-CoV-specific N protein in the brain, lungs, spleen, and intestines of infected mice. Furthermore, SADS-CoV infection triggers excessive cytokine expression that encompasses a broad array of proinflammatory mediators, including interleukin 1ß (IL-1ß), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), C-X-C motif chemokine ligand 10 (CXCL10), interferon beta (IFN-ß), IFN-γ, and IFN-λ3. This study highlights the importance of identifying neonatal mice as a model for developing vaccines or antiviral drugs against SADS-CoV infection. IMPORTANCE SADS-CoV is the documented spillover of a bat coronavirus that causes severe disease in pigs. Pigs are in frequent contact with both humans and other animals and theoretically possess a greater chance, compared to many other species, of promoting cross-species viral transmission. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Animal models are an essential feature of the vaccine design toolkit. Compared with neonatal piglets, the mouse is small, making it an economical choice for animal models for SADS-CoV vaccine design. This study showed the pathology of neonatal mice infected with SADS-CoV, which should be very useful for vaccine and antiviral studies.

Unveiling the hidden dangers: a review of non-apoptotic programmed cell death in anesthetic-induced developmental neurotoxicity.

Anesthetic-induced developmental neurotoxicity (AIDN) can arise due to various factors, among which aberrant nerve cell death is a prominent risk factor. Animal studies have reported that repeated or prolonged anesthetic exposure can cause significant neuroapoptosis in the developing brain. Lately, non-apoptotic programmed cell deaths (PCDs), characterized by inflammation and oxidative stress, have gained increasing attention. Substantial evidence suggests that non-apoptotic PCDs are essential for neuronal cell death in AIDN compared to apoptosis. This article examines relevant publications in the PubMed database until April 2024. Only original articles in English that investigated the potential manifestations of non-apoptotic PCD in AIDN were analysed. Specifically, it investigates necroptosis, pyroptosis, ferroptosis, and parthanatos, elucidating the signaling mechanisms associated with each form. Furthermore, this study explores the potential relevance of these non-apoptotic PCDs pathways to the pathological mechanisms underlying AIDN, drawing upon their distinctive characteristics. Despite the considerable challenges involved in translating fundamental scientific knowledge into clinical therapeutic interventions, this comprehensive review offers a theoretical foundation for developing innovative preventive and treatment strategies targeting non-apoptotic PCDs in the context of AIDN.

Comprehensive Subcellular Localization of Swine Acute Diarrhea Syndrome Coronavirus Proteins.

Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV was first identified in diarrheal piglets in 2017. As a novel alphacoronavirus, SADS-CoV shares ~95% identity with bat alphacoronavirus HKU2. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Thus far, no effective antiviral drugs or vaccines are available to treat infections with SADS-CoV. Therefore, knowledge of the protein-coding gene set and a subcellular localization map of SADS-CoV proteins are fundamental first steps in this endeavor. Here, all SADS-CoV genes were cloned separately into Flag-tagged plasmids, and the subcellular localizations of viral proteins, with the exception of nsp11, were detected using confocal microscopy techniques. As a result, nsp1, nsp3-N, nsp4, nsp5, nsp7, nsp8, nsp9, nsp10, nsp14, and nsp15 were localized in the cytoplasm and nuclear spaces, and these viral proteins may perform specific functions in the nucleus. All structural and accessory proteins were mainly localized in the cytoplasm. NS7a and membrane protein M colocalized with the Golgi compartment, and they may regulate the assembly of SADS-CoV virions. Maturation of SADS-CoV may occur in the late endosomes, during which envelope protein E is involved in the assembly and release of the virus. In summary, the present study demonstrates for the first time the location of all the viral proteins of SADS-CoV. These fundamental studies of SADS-CoV will promote studies of basic virology of SADS-CoV and support preventive strategies for animals with infection of SADS-CoV. IMPORTANCE SADS-CoV is the first documented spillover of a bat coronavirus that causes severe diseases in domestic animals. Our study is an in-depth annotation of the newly discovered swine coronavirus SADS-CoV genome and viral protein expression. Systematic subcellular localization of SADS-CoV proteins can have dramatic significance in revealing viral protein biological functions in the subcellular locations. Furthermore, our study promote understanding the fundamental science behind the novel swine coronavirus to pave the way for treatments and cures.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/ß Nuclear Import Pathway.

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.

Advances in lipo-solubility delivery vehicles for curcumin: bioavailability, precise targeting, possibilities and challenges.

BACKGROUND: Curcumin (Cur) is a natural pigment containing a diketone structure, which has attracted extensive attention due to its strong functional activities. However, the low solubility and poor stability of Cur limit its low bioavailability and multi-function. It is essential to develop effective measures to improve the unfavorable nature of Cur and maximize its potential benefits in nutritional intervention. SCOPE AND APPROACH: The focus of this review is to emphasize the construction of lipo-solubility delivery vehicles for Cur, including emulsion, nanoliposome and solid liposome. In addition, the potential benefits of vehicles-encapsulated Cur in the field of precise nutrition were summarized, including high targeting properties and multiple disease interventions. Further, the deficiencies and prospects of Cur encapsulated in vehicles for precise nutrition were discussed. KEY FINDINGS AND CONCLUSIONS: The well-designed lipo-solubility delivery vehicles for Cur can improve its stability in food processing and the digestion in vivo. To meet the nutritional requirements of special people for Cur-based products, the improvement of the bioavailability by using delivery vehicles will provide a theoretical basis for the precise nutrition of Cur in functional food.

Structural properties and bioavailability of curcumin were summarized.The practical problems and challenges in the utilization of curcumin were discussed.Various technologies for preparing lipo-solubility delivery vehicles for curcumin were described.The design of delivery vehicles for curcumin and intervention strategies in precise nutrition was reviewed.

A chemiluminescent magnetic microparticle immunoassay for the detection of antibody against African swine fever virus.

The p30 protein is abundantly expressed in the early stage of African swine fever virus (ASFV) infection. Thus, it is an ideal antigen candidate for serodiagnosis with the use of an immunoassay. In this study, a chemiluminescent magnetic microparticle immunoassay (CMIA) was developed for the detection of antibodies (Abs) against ASFV p30 protein in porcine serum. Purified p30 protein was coupled to magnetic beads, and the experimental conditions including concentration, temperature, incubation time, dilution ratio, buffers, and other relevant variables were evaluated and optimized. To evaluate the performance of the assay, a total of 178 pig serum samples (117 negative and 61 positive samples) were tested. According to receiver operator characteristic curve analysis, the cut-off value of the CMIA was 104,315 (area under the curve, 0.998; Youden's index, 0.974; 95% confidence interval: 99.45 to 100%). Sensitivity results showed that the dilution ratio of p30 Abs in ASFV-positive sera detected by the CMIA is much higher when compared to commercial blocking ELISA kit. Specificity testing showed that no cross-reactivity was observed with sera positive for other porcine disease viruses. The intraassay coefficient of variation (CV) was < 5%, and the interassay CV was < 10%. The p30-magnetic beads could be stored at 4 °C for more than 15 months without loss of activity. The kappa coefficient between CMIA and INGENASA blocking ELISA kit was 0.946, showing strong agreement. In conclusion, our method showed superiority with high sensitivity, specificity, reproducibility, and stability and potentialized its application in the development of a diagnostic kit for the detection of ASF in clinical samples. KEY POINTS: • ASFV tag-free p30 was successfully purified. • High sensitivity, specificity, relatively simple, and time-saving to detect antibody against ASFV were developed. • The development of CMIA will help the clinical diagnosis of ASFV and will be useful for large-scale serological test.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1 ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

TET2 is required for Type I IFN-mediated inhibition of bat-origin swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered bat-origin coronavirus with fatal pathogenicity for neonatal piglets. There is no vaccine to prevent SADS-CoV infection or clinically approved drugs targeting SADS-CoV. Therefore, unraveling cellular factors that regulate SADS-CoV for cell entry is critical to understanding the viral transmission mechanism and provides a potential therapeutic target for SADS-CoV cure. Here, we showed that Type I interferon (IFN-I) pretreatment potently blocks SADS-CoV entry into cells using lentiviral pseudo-virions as targets whose entry is driven by the SADS-CoV Spike glycoprotein. IFN-I-mediated inhibition of SADS-CoV entry and replication was dramatically impaired in the absence of TET2. These results suggest TET2 is found to serve as a checkpoint of IFN-I-meditated inhibition on the cell entry of SADS-CoV, and our discovery might constitute a novel treatment option to combat against SADS-CoV.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus P30 protein.

African swine fever virus (ASFV) causes acute, febrile, and highly contagious diseases in swine. Early diagnosis is critically important for African swine fever (ASF) prevention and control in the absence of an effective vaccine. P30 is one of the most immunogenic proteins that are produced during the early stage of an ASFV infection. This makes P30 a good serological target for ASF detection and surveillance. In this study, two P30-reactive monoclonal antibodies (mAbs), 2H2 and 5E8, were generated from mice immunized with recombinant P30 protein (rP30). Epitope mapping was performed with overlapping polypeptides, alanine mutants, and synthetic peptides. The mapping results revealed that 2H2 recognized a region located in the N-terminal, 16-48 aa. In contrast, 5E8 recognized a linear epitope in the C-terminal, 122-128 aa. Further analysis indicated that the epitope recognized by 2H2 was highly conserved in genotypes I and II, while the 5E8 epitope was conserved in most genotypes and the Ser to Pro change at position 128 in genotypes IV, V, and VI did not affect recognition. Overall, the results of this study provide valuable information on the antigenic regions of ASFV P30 and lay the foundation for the serological diagnosis of ASF and vaccine research. KEY POINTS: • Two specific and reactive mAbs were prepared and their epitopes were identified. • 2H2 recognized a novel epitope highly conserved in genotypes I and II. • 5E8 recognized a seven-amino acid linear epitope highly conserved in most genotypes.

Development and application of a colloidal-gold dual immunochromatography strip for detecting African swine fever virus antibodies.

African swine fever (ASF) is an acute and highly contagious infectious disease caused by the African swine fever virus (ASFV). Currently, there is no vaccine against ASF worldwide, and no effective treatment measures are available. For this reason, developing a simple, rapid, specific, and sensitive serological detection method for ASFV antibodies is crucial for the prevention and control of ASF. In this study, a 1:1 mixture of gold-labeled p30 and p72 probes was used as the gold-labeled antigen. The p30 and p72 proteins and their monoclonal antibodies were coated on a nitrocellulose membrane (NC) as a test (T) line and control (C) line, respectively. A colloidal-gold dual immunochromatography strip (ICS) for ASFV p30 and p72 protein antibodies was established. The results showed that the colloidal-gold dual ICS could specifically detect ASFV antibodies within 5-10 min. There was no cross-reaction after testing healthy pig serum; porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease type A virus (FMDV-A), foot-and-mouth disease type O virus (FMDV-O), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) positive sera. A positive result was obtained only for the positive control P1. The sensitivity of the test strips was 1:256, which was equivalent to that of commercially ELISA kits. Their coincidence rate with the two commercial ASFV ELISA antibodies detection kits was higher than 98%. The test strips were stably stored at 18-25 °C and 4 °C for 4 and 6 months, respectively. The colloidal-gold dual ICS prepared in this study had high sensitivity and specificity and were characterized by rapid detection, simple operation, and easy interpretation of results. Therefore, they are of great significance to diagnose, prevent, and control African swine fever. KEY POINTS: • We establish an antibody detection that is quick and can monitor an ASF infection. • We observe changes in two protein antibodies to dynamically monitor ASF infection. • We use diversified detection on a single test strip to detect both antibodies.