Despite causing fatal encephalitis and long-term neurological, cognitive, and behavioral impairments in survivors, no antiviral drugs are currently available to treat Japanese encephalitis virus (JEV) infections, highlighting the urgent need for effective therapies. Antimicrobial peptides (AMPs) exhibit broad-spectrum antibiotic activity; LL-37 is one such AMP that demonstrates a broad antiviral effect against various viruses. However, some viruses resist LL-37 and its analogues, emphasizing the importance of studying the impact of specific antimicrobial peptides on different viruses. In this study, we found that the synthetic LL-37 analogue peptide WK-13-3D has strong antiviral activity against JEV. Notably, pre-treatment or co-incubation during virus adsorption led to a noticeable reduction in infection levels in human glioblastoma (T98G) cells. Mechanistically, WK-13-3D inhibits JEV infection by directly binding to the viral particle, potentially Envelope (E) protein and preventing internalization. This peptide conferred protection against mortality in JEV-infected mice. In vivo, pre-treating JEV with WK-13-3D provided complete protection against death, compared to only 16.66% survival in the control group. Overall, these results suggest that WK-13-3D could be a promising candidate for further development as an immediate post-exposure antiviral treatment or prophylactic intervention for JEV infection.

African swine fever virus (ASFV) causes devastating disease in swine. The ongoing global pandemic, driven primarily by genotype II strains and increasingly complicated by the emergence of attenuated mutants and inter-genotypic recombinants, necessitates improved diagnostic tools and deeper understanding of viral antigenic structure. The viral phosphoprotein p30 is an immunodominant antigen expressed early during infection, making it an ideal target for diagnostics and vaccine development. In this study, three novel monoclonal antibodies (mAbs) against p30, designated 2A8 (IgG2b, κ), 2B3 (IgG2a, κ), and 13A7 (IgG1, κ), were generated, all exhibiting strong reactivity with recombinant and native p30 in ASFV-infected macrophages. Epitope mapping identified three precise linear overlapping epitopes within the immunodominant aa 76-93 region: 76EHQAQEEWNM85 (2A8), 83WNMILHVLFEE93 (2B3), and 76EHQAQEEWNMILHVLF91 (13A7), all completely conserved across genotypes I, II, and I/II recombinants. Competitive binding assays revealed mutual inhibition among the mAbs consistent with epitope overlap, with mAb 13A7 demonstrating the strongest competition with a representative ASFV-positive swine serum. Leveraging this competitive inhibition profile, a competitive ELISA was developed for ASFV antibody detection. The assay exhibited satisfactory analytical sensitivity and showed no cross-reactivity with the sera positive for other major porcine viruses, and achieved 97.38% agreement (κ = 0.941) with a commercial p72 mAb-based competitive ELISA kit when testing 344 field sera. This study identifies aa 76-93 as a highly antigenic region within p30 and provides a reliable tool for ASFV antibody detection with significant potential to enhance surveillance and control programs amid the evolving complexity of the ASFV pandemic.

African swine fever virus (ASFV) infection induces oxidative stress and produces oxidative DNA damage bases, leading to oxidative DNA base damage, including the formation of 8-oxoguanine (8-oxoG). Prompt repair of these lesions is essential to maintain genome stability. The enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) initiates the base excision repair (BER) pathway by recognizing and incising 8-oxoG, while also regulating multiple biological processes through interactions with host and viral proteins. In this study, we identified a specific interaction between the N-terminal region of ASFV DEAD-box helicase D1133L and OGG1, establishing a unique role for ASFV D1133L in DNA BER. Furthermore, we demonstrated for the first time that ASFV D1133L is a substrate for the histone acetyltransferases CBP/p300 in the nucleus. Conversely, deacetylation of D1133L by HDAC5, which predominantly occurs in the cytoplasm through its interaction with OGG1, markedly enhances OGG1 incision activity on 8-oxoG. Taken together, our findings reveal a previously unrecognized function of ASFV D1133L in promoting 8-oxoG repair by binding to OGG1 to safeguard genome integrity.

African swine fever virus (ASFV), the sole known DNA arbovirus, causes acute hemorrhagic fever in domestic pigs with case fatality rates approaching case fatality 100%. While less contagiously transmitted than viral agents like foot-and-mouth disease virus, ASFV’s epidemiological significance stems from its complex transmission ecology involving soft ticks. Ornithodoros ticks are established biological vectors, with eight species confirmed globally. However, potential vectors in China, where Ornithodoros lahorensis (O. lahorensis) is endemic and remain uncharacterized. This study tested the hypothesis that O. lahorensis serves as a competent biological vector for ASFV under laboratory conditions. We hypothesized that vector competence, defined as the ability to acquire, maintain, and transmit infectious African swine fever virus, differs between soft (O. lahorensis, Argas persicus) and hard (H. longicornis, R. sanguineus, D. silvarum) tick species, and tested this using experimental transmission assays. Results demonstrated that O. lahorensis efficiently supported transstadial persistence and transovarial transmission of ASFV. Naïve pigs exposed to infected O. lahorensis developed acute ASF with case fatality 100%. Viral replication in salivary glands, midgut, and Malpighian tubules was confirmed by qPCR and transmission electron microscopy, with no significant fitness costs to ticks. In contrast, A. persicus and all ixodid species failed to transmit. Here identifies O. lahorensis as a critical ASFV vector in China, informing targeted acaricidal control in endemic regions.

The African swine fever virus (ASFV) -encoded late structural protein pA104R is a putative histone-like protein, which is also a DNA-binding related protein required for ASFV DNA replication, transcription, and genome packaging. However, the molecular mechanism underlying pA104R-host protein interactions remain unknown. To identify proteins potentially interacting with ASFV-pA104R, a primary porcine alveolar macrophage (PAM) cDNA yeast two-hybrid library was constructed, and the pig E3 ubiquitin ligase RING-finger protein 2 (RNF2) was identified, which specifically negatively regulates the proliferation of ASFV. Mechanistically, RNF2 inhibits ASFV replication by promoting the proteasomal degradation of ASFV-pA104R through K48-linked ubiquitination at pA104R lysine 5 (K5). Further studies showed that the K5R mutation impairs the interaction between pA104R and RNF2 and antagonizes for pA104R degradation by RNF2. An ASFV mutant carrying a pA104R point mutation (ASFV CN/SC/2019 pA104R-K5R) was generated based on the ASFV CN/SC/2019 (wild-type) strain. Furthermore, our findings indicate that ASFV CN/SC/2019 pA104R-K5R enhances viral replication and virulence, potentially by increasing viral transcription and/or modulating the host immune response. Accordingly, compared with the parental strain, ASFV CN/SC/2019 pA104R-K5R was more pathogenic and severe lesions in swine. Collectively, our study identifies an intrinsic antiviral protein RNF2 that mediates ASFV CN/SC/2019 pA104R-K5 site ubiquitination emerges as a potential determinant of viral replication and pathogenicity.

Conventional immunoassays such as ELISA are widely used in serological testing, yet their reliance on multi-step workflows and labeled reagents limits diagnostic scalability and speed. Bioluminescence-based biosensors are attractive alternatives that offer high sensitivity, operational simplicity, and cost efficiency for detecting diverse analytes. Here, we present a broadly compatible bioluminescent biosensor for antibody detection based on analyte-mediated reconstitution of split-Nanoluciferase (NanoLuc). The platform utilizes engineered bifunctional probes, comprising the protein G C2 domain fused to split-NanoLuc subunits (LgBiT and SmBiT), which serve dual functions in antibody binding and signal generation. Upon immunocomplex formation with multi-epitope antigens, the probes colocalize LgBiT and SmBiT, reconstituting NanoLuc activity and producing a quantifiable bioluminescent signal. We implemented this Fc-binding split-NanoLuc complementation assay to detect antibodies against African swine fever virus (ASFV). The optimized system showed high sensitivity, a wide linear dynamic range, and no cross-reactivity with sera positive for other common swine viruses. Clinical validation exhibited 97.11

Pseudorabies virus (PRV) inflicts substantial economic losses on the global swine industry. Accurate differentiation between vaccine-derived and circulating wild-type strains is critical for effective disease control and eradication programs. However, conventional diagnostic methods often rely on costly instrumentation and entail lengthy turnaround times, thereby limiting their utility for rapid, field-deployable differential diagnosis—particularly in resource-constrained settings. In this study, we developed a rapid detection system integrating recombinase polymerase amplification (RPA) with lateral flow diagnostic strips (LFD) for the simultaneous identification of PRV wild-type and vaccine strains. The assay demonstrated high analytical specificity, reliably differentiating between PRV vaccine and wild-type strains, exhibiting no cross-reactivity with other prevalent swine pathogens. It also exhibited high sensitivity, yielding clear visual results within 20 min and achieving a limit of detection of 80 copies/µL for the PRV gE gene. Moreover, the method is operationally straightforward, requires minimal training, delivers unambiguous visual readouts, and is highly cost-effective. Collectively, the RPA-LFD platform represents a robust, field-adaptable tool for on-site surveillance of PRV field strains, offering significant potential to support large-scale screening and advance pseudorabies eradication initiatives.

Nicotinamide adenine dinucleotide (NAD+) is a crucial molecule involved in numerous interconnected metabolic processes. Due to its implication in multiple viral infection responses, maintaining NAD+ homeostasis has become a promising target for host-directed therapies. Japanese encephalitis virus (JEV) causes severe, fatal encephalitis with irreversible brain damage and long-lasting neurological deficits in survivors. However, the potential interaction between JEV infection and NAD+ metabolism remains largely unclear. In this study, we found that JEV infection dysregulates NAD+ metabolism and the expression of its pathway enzyme genes in Type I interferon (IFN-α/β) receptor-deficient (A129) mice and human glioblastoma (T98G) cells. Specifically, JEV infection altered the expression of de novo/kynurenine pathway (IDO, KATII, KMO) and salvage pathway (NAMPT, NMNATs) NAD+ biosynthetic enzymes, as well as NAD+-consuming enzymes (PARPs, SIRTs), culminating in a substantial decrease in NAD+ levels. Furthermore, NAD+ depletion and JEV production increased when salvage biosynthesis was restrained through NAMPT knockdown, but these effects were reversed by supplementing nicotinamide riboside (NR) in NAMPT knockdown T98G cells. Importantly, restoring NAD+ levels with NR supplementation as an anti-JE strategy in A129 mice reduced JEV production and improved infection outcomes. In conclusion, this study demonstrates that JEV infection disrupts NAD+ metabolism, and restoring NAD+ levels inhibits JE progression. Therefore, maintaining NAD+ homeostasis and regulating its metabolic pathway could be a promising therapeutic approach for JE.

The emergence of highly pathogenic avian influenza viruses (AIVs) poses significant threats to global public health and the poultry economy. Rapid detection methods targeting the hemagglutinin (HA) protein have become increasingly unreliable due to ongoing antigenic drift and genetic mutations. In this study, we screened two monoclonal antibodies (mAbs) against the highly conserved neuraminidase (NA) protein, which were characterized by high affinity and specificity for the N9 subtype. Using the paired mAbs 12A2 and 12F12, an NA-targeting lateral flow immunoassay (NA-LFIA) was developed for the rapid detection of N9 subtype AIVs. The NA-LFIA demonstrated exceptional specificity, exhibiting no cross-reactivity with the N1, N2, or N6 subtypes. The limits of detection (LODs) were established at 103.3 TCID50/0.1 mL for the virus and 98 ng/mL for the recombinant NA protein. The test strips exhibited excellent reproductivity for detecting the NA antigen, with intra-assay and inter-assay variations of 5.32% and 6.05%, respectively. Stability testing confirmed a six-month shelf life without any loss of sensitivity. These finds indicate that the NA-LFIA is a robust, rapid point-of-care testing (POCT) tool for the early detection of N9 subtype AIV infection.

Efficient separation of Staphylococcus aureus (S. aureus) from complex samples is crucial for sensitive detection. Herein, we fabricated a novel cefradine-modified magnetic nanoparticle (CMN) for selective capture of S. aureus. The biomolecule-free CMN could specifically capture target S. aureus due to the specific recognition between cefradine and penicillin binding proteins on the surface of S. aureus, and had the advantages of good stability, fast magnetic responsiveness, simple preparation, and low cost. Additionally, a new vancomycin-modified time-resolved fluorescent microsphere (VTFM) was prepared for electively labeling S. aureus captured by CMN. The antibody-free VTFM could selectively label S. aureus by the specific interaction between vancomycin and peptidoglycan on the surface of S. aureus, and could generate stable, sensitive, and easily quantifiable fluorescence signals. For proving the practicability, CMN linked VTFM (CMN-VTFM) strategy was proposed for the determination of S. aureus. Briefly, the CMN first selectively captured S. aureus from complex samples, and then the VTFM specifically labeled S. aureus captured by CMN; subsequently, CMN-bacteria-VTFM was separated and eluted to release the VTFM; finally, after removing CMN-bacteria, the VTFM was quantitatively analyzed. The biomolecule-free CMN-VTFM strategy with limit of detection of 92 CFU mL−1 for milk and 73 CFU mL−1 for spinach, was successfully used for detecting S. aureus in spiked real samples with recovery between 93.7 % and 100.1 % and relative standard deviation between 3.1 % and 6.1 %, respectively. Therefore, we believe that the CMN-VTFM strategy based on the antibiotic-based recognition has great potential in the field of ultrasensitive determination of pathogens.

The prevalence of tick-borne bacterial and viral diseases, which pose a serious threat to human and livestock health, is increasing worldwide. At present, only a limited number of tick-borne pathogens have been reported, and no analysis of the microbial pathogen community in ticks has been carried out. We sequenced the viral metagenome of Ornithodoros lahorensis species of ticks from the Chinese mainland and identified 390 RNA viruses with unique microbial compositions. A total of 992 assembled viral transcriptomes revealed the breadth and diversity of the genome structure of tick-borne viruses, reflecting the importance of ticks as RNA viral pools. We analyzed the phylogeny of different virus families to investigate virus evolution and found that the most diverse tick-associated viruses belonged to the family Siphoviridae, which diverged earlier in evolutionary time than other arboviruses. There were only a few tick-specific viruses, whereas the number of vertebrate-infecting viruses in ticks was greater. We hope that our virus sequencing dataset will facilitate future important research on viruses carried by ticks that can infect vertebrates.

African swine fever (ASF) is an acute and highly contagious disease that has caused great losses in the past years. It is caused by African swine fever virus (ASFV), which is a large DNA virus encoding about 165 genes. It has been shown that the purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis, and the virus utilizes apoptotic bodies for infection and cell cell transmission. The ASFV-encoded RNA polymerase subunit C315R is thought to play an important role in ASFV replication and transcription. However, its involvement in ASFV infection, particularly in host response, remains only partially understood. In this study, the role of C315R in enhancing ASFV replication was investigated through RNA-Seq transcriptomic analysis, which was based on 3D4/21 cells transfected the plasmid expressing HA-tagged C315R or the empty vector. Our findings revealed that C315R significantly upregulates the expression of inflammatory mediators with a particular emphasis on IL-6. The most differentially expressed genes (DEGs) were predominantly associated with the TNF, IL-17, MAPK, and JAK STAT signaling pathways. RNA-seq results were validated through RT-PCR. Subsequently, we observed that ASFV infection increases IL-6 expression and STAT3 phosphorylation, which is regulated by the ASFV C315R protein. Notably, inhibiting STAT3 phosphorylation with specific inhibitors suppressed ASFV replication. In conclusion, our study demonstrates that the ASFV C315R protein actives STAT3 phosphorylation through promoting the transcription of IL-6 to facilitate virus replication. These findings highlight C315R as a positive regulator in the IL-6 STAT3 signaling axis during ASFV infection.

Glucocorticoids play a key role in a variety of physiological processes, but their extensive use in the environment has brought potential health hazards. Herein, it is of great necessity to develop a rapid and efficient method for the detection of glucocorticoids. In this work, a hydrophilic core-shell structured magnetic covalent organic framework (HMCOF) was fabricated via a post-modification strategy for the efficient magnetic solid-phase extraction (MSPE) of five glucocorticoids from tap water and milk samples. The HMCOF featured a Fe₃O₄@SiO₂ magnetic core encapsulated by a porous COF shell modified with polyethylene glycol, endowing it with a hydrophilic outer layer, porous structure and sufficient paramagnetism. The adsorption studies showed that HMCOF exhibited high adsorption capacities (50.77-80.25 mg g-1) for glucocorticoids. Notably, HMCOF retained 80 % of its adsorption capacity after 5 cycles, confirming its reusability. Under the optimal conditions of MSPE, the HMCOF-based MSPE-UPLC method was developed to test five glucocorticoids, which demonstrated good linearity (5-150 ng mL-1, R2 ≥ 0.9991), low detection limits (0.2-1.4 ng mL-1) and satisfactory spiked recovery rates (89.5-114.2 %) with intra-day variability below 4.3 % and inter-day precision within 6.3 %. The method underscores the potential of HMCOF serving as an adsorbent for MSPE, providing a promising approach for the analysis of glucocorticoids within food products.

The African swine fever virus (ASFV), a complex DNA virus belonging to the Asfarviridae family, is a significant threat to the global swine industry because of its high mortality rates and impact on international trade. The establishment of a stable and efficient cell culture model of ASFV in vitro is helpful for the development of effective vaccines. Several passaged cell lines supporting ASFV replication have been reported to meet the scientific purpose of serial passage of ASFV to a certain extent, but it remains to be determined whether gene expression is lost or whether immunogenicity changes after serial passage of the virus. It is also unclear these edited cell lines how to affect ASFV replication. In our previous study, 3D4/21 cells were transduced with a lentivirus packaging system to express the BD1/2 domain of bromodomain-containing protein 4 (BRD4-BD1/2) and establish a 3D4/21-BD1/2 cell line, which efficiently increased ASFV replication. In this study, the role of bromodomain-containing protein 4 (BRD4), particularly its BD1/2 domains,in enhancing ASFV replication was investigated using an engineered 3D4/21 cell line. Through RNA-Seq transcriptomic analysis, we revealed that the host BRD4 protein facilitates ASFV infection and suppresses key transcription factors (CDK9 and p-CDK9) and inflammatory cytokine expression by downregulating transcriptional regulatory signaling pathways and suppressing innate immune responses. This dual mechanism of BRD4-BD1/2 in promoting ASFV immune evasion and adaptation underscores the virus's strategic exploitation of host epigenetic factors. These findings provide valuable insights into viral pathogenesis and identify potential therapeutic targets, paving the way for future antiviral strategies.

Emerging infectious diseases caused by various tick-borne microorganisms (TBMs) pose public and animal health concerns, including camels, with no defined global distribution. In this study, 150 blood samples and 288 ticks were collected from symptomatic two-humped camels (Camelus bactrianus) in Gaotai County, Gansu Province, China. Morphologically identified ticks were confirmed using cytochrome oxidase I (COI), and the findings revealed two species, Hyalomma asiaticum and Haemaphysalis longicornis (prevalence: 245/288 [88.19 %] and 34/288 [11.81 %], respectively). The extracted Genomic DNA from blood and ticks was processed by conventional PCR to investigate the existing TBMs based on 16S rRNA, 18S rRNA, and 17-kDa genes. Different TBMs, including Anaplasma bovis, Colpodella sp., Rickettsia rickettsii, and Candidatus Rickettsia jingxinensis, have been documented as single infections at different rates. High single infection rates (198/218; 90.83 % and 117/150; 78.00 %) of A. bovis in Hy. asiaticum and camel blood were recorded, whereas the lowest single infection rate (3/22; 13.64 %) of R. rickettsii was noted in Hae. longicornis. Co-infection with Rickettsia spp. + A. bovis (20/288; 6.94 %), Colpodella sp. + A. bovis (14/288; 4.86 %), Colpodella sp. + Rickettsia spp. (1/288; 0.35 %), and Colpodella sp. + Rickettsia spp. + A. bovis (1/288; 0.35 %) were recorded as concurrent infection. Phylogenetic analysis revealed that the representative TBMs have close similarities and clustered together with their corresponding isolates from China, South Korea, India, the USA, Mexico, Bangladesh, Malawi, Japan, Pakistan, Cyprus, Nigeria, Poland, and Brazil. These findings present a preliminary baseline regarding TBMs infection in camel blood and ticks and provide a framework for further studies on the prevalence and effective control measures for ticks and tick-associated diseases.

ABSTRACT Multigene family (MGF) 360 genes, which are African swine fever virus (ASFV) virulence genes, primarily target key host immune molecules to suppress host interferon (IFN) production and interferon-stimulated gene (ISG) transcription, impairing host innate immune responses for efficient viral replication. However, the interactions between MGF 360 virulence genes and host molecules, as well as the mechanisms through which MGF 360 genes regulate host immune responses and IFN signaling, require further elucidation. In this study, we discovered that ASFV MGF_360-4L interacts with MDA5 and recruits the mitochondrial selective autophagy receptor SQSTM1 to degrade MDA5, thus impairing IFN signaling and compromising host innate immune responses. Furthermore, MGF_360-4L inhibits the interaction between MDA5 and MAVS, blocking ISG15-mediated ISGylation of MDA5. MGF_360-4L deficiency significantly attenuated virus-induced mitochondrial autophagy in vitro. Additionally, OAS1 ubiquitinates MGF_360-4L at residues K290, K295, and K327. Finally, a recombinant ASFV lacking the MGF_360-4L gene (ASFV-∆MGF_360-4L) was generated using ASFV-CN/SC/2019 as the backbone, which demonstrated that the replication kinetics of ASFV-∆MGF_360-4L in PAM cells were like those of the highly virulent parental ASFV-WT in vitro. Domestic pigs infected with ASFV-∆MGF_360-4L exhibited milder symptoms than those infected with parental ASFV-WT, and ASFV-∆MGF_360-4L-infected pigs presented with enhanced host innate antiviral immune response, confirming that the deletion of the MGF_360-4L gene from the ASFV genome highly attenuated virulence in pigs and provided effective protection against parental ASFV challenge. In conclusion, we identified a novel ASFV virulence gene, MGF_360-4L, further elucidating ASFV infection mechanisms and providing a new candidate for vaccine development.IMPORTANCEAfrican swine fever virus (ASFV) infection causes acute death in pigs, and there is currently no effective vaccine available for prevention. Multigene family (MGF) virulence genes have been shown to be crucial for ASFV’s ability to evade host innate immune responses. However, the functions of most MGF genes remain unknown, which poses significant challenges for the development of ASFV vaccines and antiviral drugs. In this study, we identified a virulence gene of ASFV, MGF_360-4L, that targets and recruits the selective autophagy receptor p62 to mediate the degradation of the dsRNA sensor MDA5, thereby blocking interferon signaling. Additionally, it inhibits the ISG15-mediated ISGylation activation of MDA5. ASFV lacking MGF_360-4L showed reduced virulence and provided protection in pigs. Our data identify a novel virulence gene and provide new insights for ASFV vaccine development.

The bovine IgG1 Fc receptor (boFcγRIII) is a homologue to human FcγRIII (CD16) that binds bovine IgGI with medium–low affinity. In order to identify the Fc-binding site on the bovine IgG1 Fc receptor (boFcγRIII), peptides derived from the second extracellular domain (EC2) of boFcγRIII were synthesized and conjugated with the carrier protein. With a Dot-blot assay, the ability of the peptides to bind bovine IgG1 was determined, and the IgG1-binding peptide was also identified via truncation and mutation. The minimal peptide AQRVVN corresponding to the sequence 98–103 of boFcγRIII bound bovine IgG1 in Dot-blot, suggesting that it represents a linear ligand-binding site located in the putative A–B loop of the boFcγRIII EC2 domain. Mutation analysis of the peptide showed that the residues of Ala98, Gln99, Val101, Val102 and Asn103 within the Fc-binding site are critical for IgG1 binding on boFcγRIII. The functional peptide identified in this paper is of great value to the IgG–Fc interaction study and FcR-targeting drug development.

African swine fever (ASF) is a highly infectious and devastating disease that poses a significant threat to the global swine industry. The rapid spread of ASF and its ongoing pandemics continue to impact pig farming worldwide. The absence of an effective vaccine, coupled with the complexity of the African swine fever virus (ASFV), makes the control and eradication of ASF a formidable challenge. Nanobodies, derived from camelids, have emerged as promising alternatives to conventional monoclonal antibodies, offering distinct advantages in various biological applications. In this study, specific nanobodies targeting the ASFV K205R protein were selected from a phage-displayed immune library. Ten individual nanobodies were isolated based on their complementary determining regions (CDRs), and four were found to bind to the naive K205R protein of ASFV. After evaluation, nanobody VHH1 was selected for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. The assay was optimized for various reaction conditions, and the cut-off value was determined to be 26.85%, with diagnostic sensitivity and specificity of 97.52% and 97.48%, respectively. No cross-reactivity was observed with sera from pigs infected with other swine viruses, and the assay exhibited a detection sensitivity of 1:128. Comparative analysis of clinical samples showed a high concordance rate (98.98%) between the nanobody-based and monoclonal antibody-based ELISAs (Mab-cELISA). In conclusion, this study presents a phage-displayed nanobody-based competitive ELISA for the detection of ASFV antibodies, which could be valuable for ASF sero-surveillance. Additionally, the K205R-specific nanobodies identified here may be adapted for other biological or biomedical applications.

Rapid and reliable analytical techniques play important roles in various research fields and are particularly crucial for diagnosing infectious diseases in clinical settings. African swine fever (ASF) is a devastating viral pig disease for which no effective vaccine is available. The ongoing ASF pandemic has highlighted the importance of rapid and accurate diagnosis, which enables the timely implementation of control and eradication measures. In this study, a ready-to-use bioluminescence immunosensor based on a split-nanoluciferase (NanoLuc) reporter system was proposed for the one-step sensitive detection of ASF virus (ASFV) antibodies. Specifically, the NanoLuc subunits SmBiT/LgBiT were each genetically fused to the ASFV p30 protein and protein G and used as probes. The simultaneous binding of the probes to ASFV IgGs induced the reconstitution of functional NanoLuc, which can generate a strong bioluminescent signal output by catalysing the substrate furimazine. This immunosensor allows the rapid and homogeneous detection of ASFV antibodies in solution, requiring only one incubation step of 10 min. This immunosensor also has high sensitivity, high specificity, and a wide dynamic range and is particularly promising for point-of-care testing. Comparative analysis of clinical samples validated the reliability and robustness of this approach and demonstrated high consistency with enzyme-linked immunosorbent assay (ELISA) results (concordance rate: 98.71%). These results suggest that the proposed immunosensor provides an attractive alternative to conventional immunoassays and could be easily repurposed by generating specific probes for antibody detection in other diseases.