[Effects of inhibitors of DNA repair enzymes OGG1 and MTH1 on the replication of African swine fever virus].

African swine fever virus (ASFV), as a contagious viral pathogen, is responsible for the occurrence of African swine fever (ASF), a rapidly spreading and highly lethal disease. Since ASFV was introduced into China in 2018, it has been quickly spread to many provinces, which brought great challenges to the pig industry in China. Due to the limited knowledge about the pathogenesis of ASFV, neither vaccines nor antiviral drugs are available. We have found that ASFV infection can induce oxidative stress responses in cells, and DNA repair enzymes play a key role in this process. This study employed RNA interference, RT-qPCR, Western blotting, Hemadsorption (HAD), and flow cytometry to investigate the effects of the inhibitors of DNA repair enzymes OGG1 and MTH1 on ASFV replication and evaluated the anti-ASFV effects of the inhibitors. This study provides reference for the development of anti-viral drugs.

Multi-omic characterization of allele-specific regulatory variation in hybrid pigs.

Hybrid mapping is a powerful approach to efficiently identify and characterize genes regulated through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we perform a comprehensive multi-omic characterization of regulatory variation across the brain, liver, muscle, and placenta through four developmental stages. We produce one of the largest multi-omic datasets in pigs to date, including 16 whole genome sequenced individuals, as well as 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We develop a read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We show that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identify 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. We integrate methylation, chromatin accessibility, and gene expression data to show that allele specific expression can be explained in great part by allele specific methylation and/or chromatin accessibility. This study provides a comprehensive characterization of regulatory variation across multiple tissues and developmental stages in pigs.

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81 kcal/mol, while the docking energy for meropenem was - 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28 kcal/mol, compared with - 4.60 kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

An immunochromatographic strip sensor for marbofloxacin residues.

Marbofloxacin (MBF) was once widely used as a veterinary drug to control diseases in animals. MBF residues in animal food endanger human health. In the present study, an immunochromatographic strip assay (ICSA) utilizing a competitive principle was developed to rapidly detect MBF in beef samples. The 50% inhibitory concentration (IC50) and the limit of detection (LOD) of the ICSAs were 2.5 ng/mL and 0.5 ng/mL, respectively. The cross-reactivity (CR) of the MBF ICSAs to Ofloxacin (OFL), enrofloxacin (ENR), norfloxacin (NOR), and Ciprofloxacin (CIP) were 60.98%, 32.05%, 22.94%, and 23.58%, respectively. The CR for difloxacin (DIF) and sarafloxacin (SAR) was less than 0.1%. The recovery rates of MBF in spiked beef samples ranged from 82.0% to 90.4%. The intra-assay and interassay coefficients of variation (CVs) were below 10%. In addition, when the same authentic beef samples were detected in a side-by-side comparison between the ICSAs and HPLC‒MS, no statistically significant difference was observed. Therefore, the proposed ICSAs can be a useful tool for monitoring MBF residues in beef samples in a qualitative and quantitative manner.

Identification of the Linear Fc-Binding Site on the Bovine IgG1 Fc Receptor (boFcγRIII) Using Synthetic Peptides.

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.

Insight into Hyalomma anatolicum biology by comparative genomics analyses.

Hyalomma anatolicum is an obligatory blood-sucking ectoparasite and contributes to the transmission of Crimean-Congo haemorrhagic fever (CCHF) virus, Theileria spp. and Babesia spp. Progress in exploring the adaptive strategy of this ectoparasite and developing tools to fight it has been hindered by the lack of a complete genome. Herein, we assembled the genome using diverse sources of data from multiple sequencing platforms and annotated the 1.96 Gb genome of Hy. anatolicum. Comparative genome analyses and the predicted protein encoding genes reveal unique facets of this genome, including gene family expansion associated with blood feeding and digestion, multi-gene families involved in detoxification, a great number of neuropeptides and corresponding receptors regulating tick growth, development, and reproduction, and glutathione S-transferase genes playing roles in insecticide resistance and detoxification of multiple xenobiotic factors. This high quality reference genome provides fundamental data for obtaining insights into a variety of aspects of tick biology and developing novel strategies to fight notorious tick vectors of human and animal pathogens.

Composition, function, and timing: exploring the early-life gut microbiota in piglets for probiotic interventions.

BACKGROUND: The establishment of a robust gut microbiota in piglets during their early developmental stage holds the potential for long-term advantageous effects. However, the optimal timeframe for introducing probiotics to achieve this outcome remains uncertain. RESULTS: In the context of this investigation, we conducted a longitudinal assessment of the fecal microbiota of 63 piglets at three distinct pre-weaning time points. Simultaneously, we gathered vaginal and fecal samples from 23 sows. Employing 16S rRNA gene and metagenomic sequencing methodologies, we conducted a comprehensive analysis of the fluctuation patterns in microbial composition, functional capacity, interaction networks, and colonization resistance within the gut microbiota of piglets. As the piglets progressed in age, discernible modifications in intestinal microbial diversity, composition, and function were observed. A source-tracking analysis unveiled the pivotal role of fecal and vaginal microbiota derived from sows in populating the gut microbiota of neonatal piglets. By D21, the microbial interaction network displayed a more concise and efficient configuration, accompanied by enhanced colonization resistance relative to the other two time points. Moreover, we identified three strains of Ruminococcus sp. at D10 as potential candidates for improving piglets' weight gain during the weaning phase. CONCLUSIONS: The findings of this study propose that D10 represents the most opportune juncture for the introduction of external probiotic interventions during the early stages of piglet development. This investigation augments our comprehension of the microbiota dynamics in early-life of piglets and offers valuable insights for guiding forthcoming probiotic interventions.

OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Improving the accuracy of genomic prediction for meat quality traits using whole genome sequence data in pigs.

BACKGROUND: Pork quality can directly affect customer purchase tendency and meat quality traits have become valuable in modern pork production. However, genetic improvement has been slow due to high phenotyping costs. In this study, whole genome sequence (WGS) data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction (GBLUP) for meat quality in large-scale crossbred commercial pigs. RESULTS: We produced WGS data (18,695,907 SNPs and 2,106,902 INDELs exceed quality control) from 1,469 sequenced Duroc × (Landrace × Yorkshire) pigs and developed a reference panel for meat quality including meat color score, marbling score, L* (lightness), a* (redness), and b* (yellowness) of genomic prediction. The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population. Using different marker density panels derived from WGS data, accuracy differed substantially among meat quality traits, varied from 0.08 to 0.47. Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39% to 75%. We optimized the marker density and found medium- and high-density marker panels are beneficial for the estimation of heritability for meat quality. Moreover, we conducted genotype imputation from 50K chip to WGS level in the same population and found average concordance rate to exceed 95% and r2 = 0.81. CONCLUSIONS: Overall, estimation of heritability for meat quality traits can benefit from the use of WGS data. This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

Identification and Characterization of Nanobodies from a Phage Display Library and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most lethal and devastating diseases of domestic and wild swine. The continual spread and frequent outbreaks of ASF have seriously threatened the pig and pig-related industries, causing great socioeconomic losses at unprecedented proportions. Although ASF has been documented for a century, no effective vaccine or antiviral treatment is currently available. Nanobodies (Nbs) derived from heavy-chain-only antibodies in camelids have been discovered to be effective as therapeutics and robust biosensors in imaging and diagnostic applications. In the present study, a high-quality phage display library containing specific Nbs raised against ASFV proteins was successfully constructed, and 19 nanobodies specific to ASFV p30 were preliminarily identified by phage display technology. After extensive evaluation, nanobodies Nb17 and Nb30 were employed as immunosensors and applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ASFV in clinical specimens. This immunoassay showed a detection limit of approximately 1.1 ng/mL target protein and 102.5 hemadsorption (HAD50/mL) of ASFV and exhibited high specificity with no cross-reaction with the other porcine viruses tested. The performances of the newly developed assay and a commercial kit in testing 282 clinical swine samples were very similar (93.62% agreement). However, the novel sandwich Nb-ELISA showed higher sensitivity than the commercial kit when serial dilutions of ASFV-positive samples were tested. The present study describes a valuable alternative technique for the detection and surveillance of ASF in endemic regions. Furthermore, additional nanobodies specific to ASFV may be developed using the generated VHH library and employed in different biotechnology fields.

Comparative genomics reveals unique features of two Babesia motasi subspecies: Babesia motasi lintanensis and Babesia motasi hebeiensis.

Parasites of the Babesia genus are prevalent worldwide and infect a wide diversity of domestic animals and humans. Herein, using Oxford Nanopore Technology and Illumina sequencing technologies, we sequenced two Babesia subspecies, Babesia motasi lintanensis and Babesia motasi hebeiensis. We identified 3,815 one-to-one ortholog genes that are specific to ovine Babesia spp. Phylogenetic analysis reveals that the two B. motasi subspecies form a distinct clade from other piroplasmas. Consistent with their phylogenetic position, comparative genomic analysis reveals that these two ovine Babesia spp. share higher colinearity with Babesia bovis than with Babesia microti. Concerning the speciation date, B. m. lintanensis split from B. m. hebeiensis approximately 17 million years ago. Genes correlated to transcription, translation, protein modification and degradation, as well as differential/specialized gene family expansions in these two subspecies may favor adaptation to vertebrate and tick hosts. The close relationship between B. m. lintanensis and B. m. hebeiensis is underlined by a high degree of genomic synteny. Compositions of most invasion, virulence, development, and gene transcript regulation-related multigene families, including spherical body protein, variant erythrocyte surface antigen, glycosylphosphatidylinositol anchored proteins, and transcription factor Apetala 2 genes, is largely conserved, but in contrast to this conserved situation, we observe major differences in species-specific genes that may be involved in multiple functions in parasite biology. For the first time in Babesia spp., we find abundant fragments of long terminal repeat-retrotransposons in these two species. We provide fundamental information to characterize the genomes of B. m. lintanensis and B. m. hebeiensis, providing insights into the evolution of B. motasi group parasites.

Genome-wide association study for loin muscle area of commercial crossbred pigs.

OBJECTIVE: Loin muscle area (LMA) is an important target trait of pig breeding. This study aimed to identify single nucleotide polymorphisms (SNPs) and genes associated with LMA in the Duroc×(Landrace×Yorkshire) crossbred pigs (DLY). METHODS: A genome-wide association study was performed using the Illumina 50K chip to map the genetic marker and genes associated with LMA in 511 DLY pigs (255 boars and 256 sows). RESULTS: After quality control, we detected 35,426 SNPs, including six SNPs significantly associated with LMA in pigs, with MARC0094338 and ASGA0072817 being the two key SNPs responsible for 1.77% and 2.48% of the phenotypic variance of LMA, respectively. Based on previous research, we determined two candidate genes (growth hormone receptor [GHR] and 3-oxoacid Co A-transferase 1 [OXCT1]) that are associated with fat deposition and muscle growth and found further additional genes (MYOCD, ARHGAP44, ELAC2, MAP2K4, FBXO4, FBLL1, RARS1, SLIT3, and RANK3) that are presumed to have an effect on LMA. CONCLUSION: This study contributes to the identification of the mutation that underlies quantitative trait loci associated with LMA and to future pig breeding programs based on marker-assisted selection. Further studies are needed to elucidate the role of the identified candidate genes in the physiological processes involved in LMA regulation.

OGG1 inhibition suppresses African swine fever virus replication.

African swine fever virus (ASFV) is an important pathogen that causes a highly contagious and lethal disease in swine, for which neither a vaccine nor treatment is available. The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises the oxidative base lesion 8-oxo-7,8-dihydroguanine (8-oxoG), has been linked to the pathogenesis of different diseases associated with viral infections. However, the role of OGG1-base excision repair (BER) in ASFV infection has been poorly investigated. Our study aimed to characterize the alteration of host reactive oxygen species (ROS) and OGG1 and to analyse the role of OGG1 in ASFV infection. We found that ASFV infection induced high levels and dynamic changes in ROS and 8-oxoG and consistently increased the expression of OGG1. Viral yield, transcription level, and protein synthesis were reduced in ASFV-infected primary alveolar macrophages (PAMs) treated by TH5487 or SU0268 inhibiting OGG1. The expression of BER pathway associated proteins of ASFV was also suppressed in OGG1-inhibited PAMs. Furthermore, OGG1 was found to negatively regulate interferon ß (IFN-ß) production during ASFV infection and IFN-ß could be activated by OGG1 inhibition with TH5487 and SU0268, which blocked OGG1 binding to 8-oxoG. Additionally, the interaction of OGG1 with viral MGF360-14-L protein could disturb IFN-ß production to further affect ASFV replication. These results suggest that OGG1 plays the crucial role in successful viral infection and OGG1 inhibitors SU0268 or TH5487 could be used as antiviral agents for ASFV infection.

Multiple-probe-assisted DNA capture and amplification for high-throughput African swine fever virus detection.

African swine fever (ASF) is one of the most devastating infectious diseases affecting domestic pigs and wild boar. The grave socio-economic impact of African swine fever infection at a global level makes large-scale rapid and robust diagnosis a critical step towards effective control. Here, we describe multiple-probe-assisted DNA capture and amplification technology (MADCAT) - a novel, sensitive, simple, and high-throughput method for detecting ASFV directly from whole blood or other complex matrices. Through a unique DNA capture approach which specifically captures the target DNA onto 96-well plate for subsequent amplification, MADCAT abandons the complicated extraction protocol and achieves ultrafast and high-throughput detection. The sample-to-result time for 96 samples is about 90 min, as compared with the 3-4 h time of the conventional real-time qPCR method. The limit of detection (LOD) of MADCAT is 0.5 copies/µL blood and is 5 times more sensitive than an extraction-based qPCR assay when testing serially diluted whole blood samples. The assay is 100% specific against other common swine pathogens. In the clinical diagnosis of 96 field samples, all 22 positive samples were correctly identified with lower Ct values than extraction-based qPCR, confirming its high diagnostic sensitivity (100%). Owing to its high-throughput, specific high sensitivity, and direct detection features, MADCAT shows great potential for use in large-scale ASFV surveillance and monitoring for effective disease control. KEY POINTS: • No nucleic acid extraction, 100% capture efficiency, and high-throughput • Ultra-high sensitivity of 0.5 DNA copies/µL or 6 DNA copies/reaction • The sample-to-answer time for 96 samples is about 90 min.

Genome-Wide Association Studies for Flesh Color and Intramuscular Fat in (Duroc × Landrace × Large White) Crossbred Commercial Pigs.

The intuitive impression of pork is extremely important in terms of whether consumers are enthusiastic about purchasing it. Flesh color and intramuscular fat (IMF) are indispensable indicators in meat quality assessment. In this study, we determined the flesh color and intramuscular fat at 45 min and 12 h after slaughter (45 mFC, 45 mIMF, 12 hFC, and 12 hIMF) of 1518 commercial Duroc × Landrace × Large White (DLY) pigs. We performed a single nucleotide polymorphism (SNP) genome-wide association study (GWAS) analysis with 28,066 SNPs. This experiment found that the correlation between 45 mFC and 12 hFC was 0.343. The correlation between 45 mIMF and 12 hIMF was 0.238. The heritability of the traits 45 mFC, 12 hFC, 45 mIMF, and 12 hIMF was 0.112, 0.217, 0.139, and 0.178, respectively, and we identified seven SNPs for flesh color and three SNPs for IMF. Finally, several candidate genes regulating these four traits were identified. Three candidate genes related to flesh color were provided: SNCAIP and PRR16 on SSC2, ST3GAL4 on SSC5, and GALR1 on SSC1. A total of three candidate genes related to intramuscular fat were found, including ABLIM3 on SSC2, DPH5 on SSC4, and DOCK10 on SSC15. Furthermore, GO and KEGG analysis revealed that these genes are involved in the regulation of apoptosis and are implicated in functions such as pigmentation and skeletal muscle metabolism. This study applied GWAS to analyze the scoring results of flesh color and IMF in different time periods, and it further revealed the genetic structure of flesh color and IMF traits, which may provide important genetic loci for the subsequent improvement of pig meat quality traits.

An improved luciferase immunosorbent assay for ultrasensitive detection of antibodies against African swine fever virus.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal infectious disease of pigs and causes great socioeconomic losses globally. The reliable diagnostic method is critical for prevention and control of the disease. In this study, an improved Luciferase immunosorbent assay (LISA) for detecting ASF was developed using the cell lysates containing ASFV p35 protein fused with a reporter Nano-luciferase (p35-Luc protein). The improved method avoids the complicate procedures of immobilizing the serum samples with protein G in the normal LISA method, and replaced by directly coating the serum samples with carbonate buffer, therefore reduces the productive cost and simplifies the operation procedures. The p35-Luc LISA exhibited high specificity for anti-ASFV sera while no cross-reactions with the sera against other swine viruses. The detection limit of the p35-Luc LISA was shown to be at least four times higher than that of the p35 based indirect ELISA established in our lab. The receiver operating characteristic (ROC) analysis showed the 96.36% relative specificity and 96.97% relative sensitivity of the p35-Luc LISA with the cutoff values of 3.55 as compared to the commercial Ingezim p72-ELISA kit. Furthermore, a total of 248 serum samples were tested by both the p35-Luc LISA and commercial Ingezim p72-ELISA kit, and there was a high degree of agreement (97.6%, kappa = 0.9753) in the performance of the two assays. Collectively, the improved LISA based on the p35-Luc protein could be used as a rapid, ultrasensitive, cost-effective and reliable diagnostic tool for serological survey of ASF in pig farms.

Identification and characterization of nanobodies specifically against African swine fever virus major capsid protein p72.

African swine fever virus (ASFV) is a large and very complex DNA virus. The major capsid protein p72 is the most predominant structural protein and constitutes the outmost icosahedral capsid of the virion. In the present study, the nanobodies against ASFV p72 protein were screened from a camelid immune VHH library by phage display technique. Nine distinct nanobodies were identified according to the amino acid sequences of the complementary determining regions (CDRs), and contain typical amino acid substitutions in the framework region 2 (FR2). Six nanobodies were successfully expressed in E. coli, and their specificity and affinity to p72 protein were further evaluated. The results showed that nanobodies Nb25 had the best affinity to both recombinant and native p72 protein of ASFV. The Nb25 possesses an extremely long CDR3 with 23 amino acids compared with other nanobodies, which may allow this nanobody to access the hidden epitopes of target antigen. Furthermore, the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV in a sandwich immunoassay, and its application as a biosensor to target virus in PAM cells was verified by an immunofluorescence assay. Nanobodies have been proven to possess many favorable properties with small size, high affinity and specificity, easier to produce, low costs and deep tissue penetration that make them suitable for various biotechnological applications. These findings suggest that nanobody Nb25 identified herein could be a valuable alternative tool and has potential applications in diagnostic and basic research on ASFV.

Development of a highly sensitive Gaussia luciferase immunoprecipitation assay for the detection of antibodies against African swine fever virus.

In recent years, African swine fever (ASF) has caused a devastating blow to the swine industry globally. Since no effective vaccine is available, strict biosafety measures and rapid diagnosis are the most effective strategies for ASF control. ASFV p30 is one of the most antigenic viral proteins that have been widely used in the field for serological diagnosis of ASF infection. In this study, we developed a luciferase immunoprecipitation system (LIPS) assay for the detection of ASFV antibodies in pig serum using Gaussia luciferase (GLuc)-tagged p30 as a diagnostic antigen. The optimal GLuc-p30 input of 107 luminance units (LU) and optimal serum dilution factor of 1/100 were set to achieve the highest P/N ratio. Based on 87 ASFV-positive and negative pig sera, the cutoff value of the S/N ratio could be set between 2.298 and 30.59 to achieve 100% sensitivity and 100% specificity. Moreover, the diagnostic sensitivity of this LIPS is comparable to that of a commercial enzyme-linked immunosorbent assay (ELISA) and the specificity of LIPS is even superior to the tested ELISA. In conclusion, we have established a LIPS assay for ASFV antibody detection, which could be a potential method for ASFV diagnosis in laboratories and farms.