African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum. Key points • We successfully screened a specific, high affinity nanobody against ASFV p54 protein. • We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system. • We establish a nanobody cELISA detection method that can monitor an ASF infection. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11981-4.

Background African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects. Results The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98). Conclusions The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1–69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

Background: African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results: Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cut-off value for the cELISA was 15.78%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The specificity of the cELISA was 98.97%, and the limit of detection was 1:320 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.56%. Conclusions: A specific, sensitive and repeatable cELISA was successfully developed based on the unique Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.