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Background Akabane virus (AKAV) is divided into five genogroups (I to V), and strains of different genogroups exhibit marked differences in pathogenicity. We isolated a genogroup II AKAV strain, TJ2016, in China in 2016, but its virulence remains unknown. The pathogenic potential of other genogroup II strains isolated in China also remains uncharacterized. The objectives of this study were to determine the pathogenicity of TJ2016. Methods Kunming or Balb/c mice at 7 days or 8 weeks of age were inoculated with TJ2016 by intracerebral (IC), intraperitoneal (IP), subcutaneous (SC), or intramuscular (IM) routes. Clinical signs, pathological alterations, and AKAV distributions in the inoculated mice were monitored and analyzed. Results Virus inoculations by the IC route resulted in 75% ~ 100% mortality of the inoculated mice regardless of the mouse strains or ages. Virus inoculations by the IP route killed 75% to 100% of the suckling mice but killed no adult mice. All the mice inoculated via SC and IM routes survived until the end of the trial. AKAV was detected only in the brains of the mice that died or were euthanized before the end of the experiment. The AKAV antigens were only identifiable within neuronal cells. Brain lesions such as proliferation and infiltration of microglial cells, perivascular cuffing (PVC) of lymphocytes and macrophages, neuronal degeneration/necrosis, vascular dilatation and congestion, etc., were observed only in the mice that died or were euthanized before the end of the experiment. Conclusions We characterized the virulence of TJ2016 by inoculating suckling and adult mice via different routes and established experimental mouse models, which holds significant implications for vaccine/drug development and further research on viral pathogenesis.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

Peatlands play an important role in the global carbon cycle. However, the initiation and development of permafrost peatlands and their responses to climate change remain unclear, hindering our understanding of the past and future of this region. In this study, we reconstructed the evolution of permafrost peatlands in the Greater Khingan Mountains (GKM) of Northeast China since 3500 cal yr BP using palynological evidence from permafrost peatland core and AMS14C dating. The results indicated that from 3500 to 2900 cal yr BP, the vegetation mainly composed of Pinus, thermophilic broad‐leaved trees, and Polypodiaceae, with a warm and wet climate constituting the peatland incubation period. From 2900 to 2250 cal yr BP, the vegetation mainly composed of Pinus, thermophilic broad‐leaved trees, and Artemisia, with a peatland initiation period characterized by a warm and humid climate. From 2250 to 1650 cal yr BP, the vegetation mainly composed of Pinus, Betula, and Polypodiaceae, with a cold and wet climate allowing peatland to flourish. From 1650 to 750 cal yr BP, the vegetation mainly composed of Pinus and Artemisia, and a dry, cold climate led to a slowdown or stagnation in peatland development. Later in this period, a warmer and wetter climate allowed the peatland to develop again, thereby completing the transition from eutrophic to mesotrophic state. Since 750 cal yr BP, the vegetation mainly composed of Pinus and Cyperaceae, indicating a colder and wetter climate allowing the peatland to flourish again, and peatlands began to change to oligotrophic state. Our results showed that the evolution of the GKM permafrost peatlands is mainly influenced by climate, and permafrost peatland development in the future will depend on trends in global climate. Pollen‐based analyses, the vegetation and climate of the Greater Khingan Mountains permafrost peatland in Northeast China were reconstructed. Cold and wet climatic environment would have facilitated the accumulation and vigorous development. Climate change and the resulting changes in the permafrost environment are the main drivers of the evolution of permafrost peatlands.

Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Accurate differentiation between Coilia brachygnathus and Coilia nasus is imperative for the effective management of fisheries, the conservation of aquatic ecosystems, and the mitigation of commercial fraud. Current morphological identification remains challenging due to their high morphological similarity—particularly for processed samples—while conventional molecular methods often lack the speed or specificity required for field applications or high-throughput screening. In this study, a novel integrated approach was developed and validated, combining TaqMan quantitative real-time PCR (qPCR). for precise genotyping of C. brachygnathus and C. nasus with Recombinase Polymerase Amplification coupled with Lateral Flow Dipstick (RPA-LFD) for rapid on-site screening. First, species-specific RPA-LFD assays were designed to target the mitochondrial COI gene sequence. This enabled visual detection within 10 min at 37 °C, with a sensitivity of 102 copies/μL, and required no complex equipment. A dual TaqMan MGB qPCR assay was further developed by validating stable differentiating SNPs (chr21:3798155, C/T) between C. brachygnathus and C. nasus, using FAM/VIC dual-labeled MGB probes. Results showed that this assay could distinguish the two species in a single tube: for C. brachygnathus, Ct values in the FAM channel were significantly earlier than those in the VIC channel (ΔCt ≥ 1), with a FAM detection limit of 125 copies/reaction; for C. nasus, only VIC channel amplification was observed, with a detection limit as low as 12.5 copies/reaction. Validation with 171 known tissue samples demonstrated 100% concordance with expected species identities. This integrated approach effectively combines the high accuracy and quantitative capacity of TaqMan qPCR for confirmatory laboratory genotyping with the speed, simplicity, and portability of RPA-LFD for initial field or point-of-need screening. This reliable, efficient, and user-friendly technique provides a powerful tool for resource management, biodiversity monitoring, and ensuring the authenticity of high-quality C. brachygnathus and C. nasus.

Climate change poses an increasing threat to livestock reproduction, with heat stress (HS) known to significantly impair ovarian function. This study aimed to elucidate the impact of HS on ovarian function and circRNA expression profiles in Hu sheep. Twelve ewes were randomly assigned to a control (Con, n = 6) or HS group (n = 6) and exposed to different temperatures for 68 days. Compared with the Con group, HS significantly increased the respiratory rate (108.33 ± 3.72 vs. 63.58 ± 2.42 breaths/min), pulse rate (121.17 ± 3.98 vs. 78.08 ± 3.31 beats/min), and rectal temperature (40.17 ± 0.14 °C vs. 39.02 ± 0.21 °C; p < 0.05). Concurrently, serum antioxidant levels were markedly decreased, including total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) (p < 0.05). Histological analysis revealed a significant reduction in the numbers of primordial, primary, secondary, and mature follicles, alongside an increase in antral follicles (p < 0.05). TUNEL staining demonstrated enhanced granulosa cell apoptosis (p < 0.05), accompanied by the upregulation of pro-apoptotic genes Bax and Caspase-3 and downregulation of the anti-apoptotic gene Bcl-2, as confirmed by qPCR (p < 0.05). CircRNA sequencing identified 152 differentially expressed circRNAs (120 upregulated, 32 downregulated), and enrichment analyses indicated their involvement in apoptosis, mitophagy, and the FoxO signaling pathway. Collectively, these findings demonstrate that HS impairs ovarian physiology and antioxidant defense, induces follicular damage and cell apoptosis, and alters circRNA expression profiles, providing new insights into the molecular mechanisms underlying HS-induced reproductive dysfunction in Hu sheep.

Pigs are susceptible to the deadly infectious disease known as African swine fever (ASF), which is brought on by the African swine fever virus (ASFV). As such, prompt and precise disease detection is essential. Deletion of the virulence-related genes MGF-505/360 and EP402R generated from the virulent genotype II virus significantly reduces its virulence, and animal tests using one of the recombinant viruses show great lethality and transmissibility in pigs. The isothermal technique known as recombinase polymerase amplification (RPA) is perfect for rapid in-field detection. To accurately identify ASFV MGF-505R gene-deleted mutants and assess the complex infection situation of ASF, RPA assays in conjunction with real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA-LFD assay) were created. These innovative methods allow for the direct detection of ASFV from pigs, offering in-field pathogen detection, timely disease management, and satisfying animal quarantine requirements. The specific primers and probes were designed against conserved regions of ASFV B646L and MGF-505R genes. Using recombinant plasmid DNA containing ASFV MGF-505R gene-deleted mutants as a template, the sensitivity of both ASF real-time RPA and ASF RPA-LFD assays were demonstrated to be 10 copies per reaction within 20 min at 37 °C. Neither assay had cross-reactions with CSFV, PRRSV, PPV, PRV, ot PCV2, common viruses seen in pigs, indicating that these methods were highly specific for ASFV. The evaluation of the performance of ASFV real-time RPA and ASFV RPA-LFD assays with clinical samples (n = 453) demonstrated their ability to specifically detect ASFV or MGF-505R gene-deleted mutants in samples of pig feces, ham, fresh pork, and blood. Both assays exhibited the same diagnostic rate as the WOAH-recommended real-time fluorescence PCR, highlighting their reliability and validity. These assays offer a simple, cost-effective, rapid, and sensitive method for on-site identification of ASFV MGF-505R gene-deleted mutants. As a promising alternative to real-time PCR, they have the potential to significantly enhance the prevention and control of ASF in field settings.

NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. These functions rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions. To gain further insight into the role of NS1, a tandem affinity purification (TAP) method was utilized to find unknown interaction partner of NS1. The protein complexes of NS1 and its interacting partner were purified from A549 cell using TAP-tagged NS1 as bait, and co-purified cellular factors were identified by mass spectrometry (MS). We identified cellular β-tubulin as a novel interaction partner of NS1. The RNA-binding domain of NS1 interacts with β-tubulin through its RNA-binding domain, as judged by a glutathione S-transferase (GST) pull-down assay with the GST-fused functional domains of NS1. Immunofluorescence analysis further revealed that NS1 with β-tubulin co-localized in the nucleus. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1-transfected A549 cells. Our findings suggest that influenza A virus may utilize its NS1 protein to interact with cellular β-tubulin to further disrupt normal cell division and induce apoptosis. Future work will illustrate whether this interaction is uniquely specific to the 2009 pandemic H1N1 virus.

Background Brucellosis is a serious zoonosis disease that frequently causes significant economic loss in animal husbandry and threatens human health. Therefore, we established a rapid, accurate, simple and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) for detection the antibodies of Brucella infection animals serum. Results The test strips were successfully prepared by quantum dot fluorescent microspheres (QDFM) as tracers, which were covalently coupled to an outer membrane protein of Brucella OMP22. The outer membrane protein OMP28 and monoclonal antibodies of OMP22 were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (H T / H C ) is 0.0492. The repeatability was excellent with an overall average CV of 8.78%. Under optimum conditions, the limit of detection was 1.05 ng/mL (1:512 dilution). With regard to the detection of brucellosis in 150 clinical samples, the total coincidence rate of ICST and Rose Bengal plate test (RBPT) was 97.3%, the coincidence rate of positive samples was 98.8%, the coincidence rate of negative samples was 95.3%, the sensitivity of RBPT is 1:32, and no cross reaction with the sera of other related diseases was observed. Conclusion In our present study, the QDFM has promising application for on-site screening of brucellosis owing to its high detection speed, high sensitivity, high specificity and low cost.

Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. The nucleocapsid (N) protein of AKAV possesses multiple important functions in the virus life cycle, and it is an ideal choice for AKAV detection. In this study, we successfully constructed two stable BHK-21 cell lines (C8H2 and F7E5) that constitutively express the AKAV N protein using a lentivirus system combined with puromycin selection. RT-PCR analysis confirmed that the AKAV N gene was integrated into the BHK-21 cell genome and consistently transcribed. Indirect immunofluorescence (IFA) and Western blot (WB) assays proved that both C8H2 and F7E5 cells could react with the AKAV N protein mAb specifically, indicating potential applications in AKAV detection. Furthermore, we analyzed the growth kinetics of AKAV in the C8H2 and F7E5 cell lines and observed temporary inhibition of viral replication at 12, 24 and 36 h postinfection (hpi) compared to BHK-21 cells. Subsequent investigations suggested that the reduced viral replication was linked to the down-regulation of the viral mRNAs (Gc and RdRp). In summary, we have established materials for detecting AKAV and gained new insights into the function of the AKAV N protein.