Verification and Analysis of Filter Paper-Based Intracellular Delivery of Exogenous Substances.

The intracellular delivery of exogenous substances is an essential technical means in the field of biomedical research, including cell therapy and gene editing. Although many delivery technologies and strategies are present, each technique has its own limitations. The delivery cost is usually a major limiting factor for general laboratories. In addition, simplifying the operation process and shortening the delivery time are key challenges. Here, we develop a filter paper-syringe (FPS) delivery method, a new type of cell permeation approach based on filter paper. The cells in a syringe are forced to pass through the filter paper quickly. During this process, external pressure forces the cells to collide and squeeze with the fiber matrix of the filter paper, causing the cells to deform rapidly, thereby enhancing the permeability of the cell membrane and realizing the delivery of exogenous substances. Moreover, the large gap between the fiber networks of filter paper can prevent the cells from bearing high pressure, thus maintaining high cell vitality. Results showed that the slow-speed filter paper used can realize efficient intracellular delivery of various exogenous substances, especially small molecular substances (e.g., 3-5 kDa dextran and siRNA). Meanwhile, we found that the FPS method not only does not require a lengthy operating step compared with the widely used liposomal delivery of siRNA but also that the delivery efficiency is similar. In conclusion, the FPS approach is a simple, easy-to-operate, and fast (about 2 s) delivery method and may be an attractive alternative to membrane destruction-based transfection.

Mitochondrial genome of Theileria uilenbergi endemic in sheep and goats in China.

Mitochondrial genomes provide new insights that help elucidating biological features, genetic evolution, and classification of protozoans. Theileria uilenbergi (T. uilenbergi), transmitted by Haemaphysalis qinghaiensis and H. longicornis, is considered as highly pathogenic to sheep and goats in China. This study reports and outlines features of its mitochondrial genome. The T. uilenbergi mitochondrial genome is a linear monomeric molecule of 6.0 kb length, which encodes three protein-coding genes named cytochrome c oxidase I (cox1), cytochrome b (cob), and cytochrome c oxidase III (cox3), as well as six large subunit (LSU) rRNA gene fragments, and ends in terminal inverted repeats (TIRs). The array structure and organization of the mitochondrial genome of T. uilenbergi is identical to that of T. parva. Phylogenetic analysis based on the amino acid sequences of cox1, cob, and cox3 genes suggests that T. uilenbergi is distantly related to the group of transforming Theileria species such as T. parva. This study contributes to a comprehensive understanding of the phylogeny and evolution of the mitochondrial genome of piroplasms and provides useful information of diagnostic marker for T. uilenbergi.

Comparative evaluation of two informative markers in the phylogeny of Babesia and Theileria parasites.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. In the study, we assessed the relative resolution capabilities of the DNA sequences of the nuclear genes 40S ribosomal protein S5 (RPS5) and mitochondrial DNA Cytochrome c oxidase subunit III (cox3) gene in the phylogeny of Babesia and Theileria species isolates. We demonstrated that by using the cox3 gene can recover a better supported species tree for some Theileria species than when using the nuclear RPS5 gene alone, it tends to intra-specific diversity and considerable inter-specific difference. Additionally, the combined DNA sequences of the nuclear RPS5 and cox3 gene improved the inference of evolutionary relationships among Babesia and Theileria species. The mitochondrial cox3 gene outperforms nuclear RPS5 gene and yields better resolution on the intra-specific diversity of Babesia and Theileria species. However, the combined RPS5 nuclear DNA and cox3 DNA tree had more advantage in the phylogeny of Babesia and Theileria species than that of single gene alone.

Transcriptome analysis of responses to bluetongue virus infection in Aedes albopictus cells.

BACKGROUND: Bluetongue virus (BTV) causes a disease among wild and domesticated ruminants which is not contagious, but which is transmitted by biting midges of the Culicoides species. BTV can induce an intense cytopathic effect (CPE) in mammalian cells after infection, although Culicoides- or mosquito-derived cell cultures cause non-lytic infection with BTV without CPE. However, little is known about the transcriptome changes in Aedes albopictus cells infected with BTV. METHODS: Transcriptome sequencing was used to identify the expression pattern of mRNA transcripts in A. albopictus cells infected with BTV, given the absence of the Culicoides genome sequence. Bioinformatics analyses were performed to examine the biological functions of the differentially expressed genes. Subsequently, quantitative reverse transcription-polymerase chain reaction was utilized to validate the sequencing data. RESULTS: In total, 51,850,205 raw reads were generated from the BTV infection group and 51,852,293 from the control group. A total of 5769 unigenes were common to both groups; only 779 unigenes existed exclusively in the infection group and 607 in the control group. In total, 380 differentially expressed genes were identified, 362 of which were up-regulated and 18 of which were down-regulated. Bioinformatics analyses revealed that the differentially expressed genes mainly participated in endocytosis, FoxO, MAPK, dorso-ventral axis formation, insulin resistance, Hippo, and JAK-STAT signaling pathways. CONCLUSION: This study represents the first attempt to investigate transcriptome-wide dysregulation in A. albopictus cells infected with BTV. The understanding of BTV pathogenesis and virus-vector interaction will be improved by global transcriptome profiling.

Complete genome sequence of a bovine ephemeral fever virus JT02L strain in mainland China.

In this study, we report the complete genome sequence of bovine ephemeral fever virus (BEFV) JT02L, which has been used in our laboratory, in mainland China, for more than a decade. The genome is 14941 nucleotide (nt), comprising a leader sequence of 50 nt, nucleoprotein (N) gene of 1328 nt, phosphoprotein (P) gene of 858 nt, matrix protein (M) gene of 691 nt, glycoprotein (G) gene of 1897 nt, non-structural glycoprotein (GNS) gene of 1785 nt, α1α2 gene of 638 nt, ß gene of 460 nt, γ gene of 400 nt, large multi-functional enzyme (L) gene of 6470 nt and a trailer sequence of 73 nt. Individual genes are separated by intergenic regions (IGRs) of 26, 44, 47, 51, 37, 39, 68 and -21 nt respectively. The overall organization is similar to an Australian BEFV isolate BB7721 but demonstrates some distinctive features including longer α3 and ß open reading frames, intact termination/polyadenylation (TTP) sequence downstream of the ß open reading frame and a longer ß-γ IGR integrated with a 38 nt AT-rich fragment. To our knowledge, this is the first report describing the complete genome of a BEFV strain of East Asian lineage, which may facilitate studies on genomic diversity among geographic strains of BEFV in China and the world.

Proteomic analysis of sheep primary testicular cells infected with bluetongue virus.

Bluetongue virus (BTV) causes a non-contagious, arthropod-transmitted disease in wild and domestic ruminants, such as sheep. In this study, we used iTRAQ labeling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in BTV-infected sheep testicular (ST) cells. Relative quantitative data were obtained for 4455 proteins in BTV- and mock-infected ST cells, among which 101 and 479 proteins were differentially expressed at 24 and 48 h post-infection, respectively, indicating further proteomic changes during the later stages of infection. Ten corresponding genes of differentially expressed proteins were validated via real-time RT-PCR. Expression levels of three representative proteins, eIF4a1, STAT1 and HSP27, were further confirmed via western blot analysis. Bioinformatics analysis disclosed that the differentially expressed proteins are primarily involved in biological processes related to innate immune response, signal transduction, nucleocytoplasmic transport, transcription and apoptosis. Several upregulated proteins were associated with the RIG-I-like receptor signaling pathway and endocytosis. To our knowledge, this study represents the first attempt to investigate proteome-wide dysregulation in BTV-infected cells with the aid of quantitative proteomics. Our collective results not only enhance understanding of the host response to BTV infection but also highlight multiple potential targets for the development of antiviral agents.

Genome analysis of an atypical bovine pestivirus from fetal bovine serum.

We report the complete genome sequence of a bovine pestivirus LVRI/cont-1 originated from a commercial batch of fetal bovine serum. Its complete genome consists of 12,282 nucleotides (nt), which contain an open reading frame (ORF) of 11,700 bp flanked by 5' and 3' untranslated regions (383 and 199 bp). The size of the 5'UTR and the individual protein coding region of LVRI/cont-1 are identical to those of the reference virus Th/04_KhonKaen, but it has a deletion of the first 56 nt in the 3'UTR. Alignment of the complete nucleotide sequence and phylogenetic analysis indicate that this viral isolate belongs to atypical pestiviruses.

Trafficking of bluetongue virus visualized by recovery of tetracysteine-tagged virion particles.

UNLABELLED: Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses. IMPORTANCE: Live-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

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.

Foot-and-mouth disease virus carrier status in Bos grunniens yaks.

BACKGROUND: The carrier status of foot-and-mouth disease virus (FMDV) is complicated, and the role of carrier animals in virus transmission is controversial. To investigate the carrier status of FMDV in animals that live in high altitude, Bos grunniens yaks were infected experimentally with FMDV O/Akesu/58. RESULTS: All of the yaks showed clinical signs of foot-and-mouth disease (FMD). Total antibody levels against FMDV measured by liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) and antibody levels against nonstructural proteins (NSP) showed dynamic changes. Three of the five yaks were indentified as carrier animals by RT-PCR method, and the OP fluids from carrier yaks can cause cytopathic effect (CPE) on BHK-21 cells. At last, five persistent infection strains were isolated. Nucleotide mutations of VP1 gene were analyzed. CONCLUSIONS: After infected with FMDV, all of the yaks showed typical clinical signs. Yaks can keep carrier status for at least 8 months. Total antibody levels against FMDV measured by LPB-ELISA and antibody levels against NSP were at high level for carrier yaks. Sequence alignment of the five isolated strains showed obvious gene and protein mutations.

Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice.

Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.

Characterization of a bovine viral diarrhea virus originated from cattle in Gansu Province, China.

A bovine viral diarrhea disease virus (BVDV) GS-4 was isolated in Western China form dairy cattle with respiratory disease. Genomic comparison analysis with the 5' half genome sequence encompassing the coding region of N(pro), capsid, and envelope glycoproteins showed that the GS-4 should be classified into BVDV-1b1, which is considered as one of the predominant subgenotypes found in China. This classification was confirmed by phylogenetic analysis based on E2 coding region.

The GTPase activity and isoprenylation of Swine GBP1 are critical for inhibiting the production of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is a flavivirus that cause severe neurological deficits. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on many RNA and DNA viruses via diverse mechanisms, however, the roles and the action modes of GBP1 in the antiviral effect on the production of JEV RNA and infectious virions remain to be clarified. In this study, we found that the RNA levels of swine GBP1 (sGBP1) in PK15 cells were up-regulated at the late stage of JEV infection. The overexpression of sGBP1 significantly inhibited the production of JEV while the knockdown of sGBP1 promoted the production of JEV. The GTPase activity and isoprenylation of sGBP1 both are critical for anti-JEV activity. The GTPase activity of sGBP1 is responsible for inhibiting the production of JEV genomic RNA. The isoprenylation of sGBP1 inhibited the expression and cleavage of JEV prM to decrease the yields of infectious virions, which may be associated with the interaction between sGBP1 and cellular proprotein convertase furin. Taken together, the study dissected the action modes of sGBP1with potent anti-JEV activity in more details.

Interferon-stimulated gene 15 facilitates BTV replication through interacting with the NS1 protein.

Bluetongue virus (BTV) infection effectively activates the innate immune response, followed by the expression of interferon (IFN) and multiple interferon-stimulated genes (ISGs). ISG15 is one of the most induced ISGs, and often plays a role in inhibiting virus replication. This study aims to explore the role and specific mechanisms of ovine ISG15 (oISG15) in BTV infection. We found that the transcription level of oISG15 was upregulated in a time-dependent and BTV multiplicity of infection-dependent manner. The overexpression of exogenous oISG15 enhances BTV replication, whereas the knockdown of endogenous oISG15 inhibits BTV replication. The viral protein in wild-type oISG15-overexpressed cells and ISGylation defective oISG15-overexpressed cells have no significant differences, which indicated that oISG15 promoted BTV replication in an ISGylation-independent manner. A co-immunoprecipitation assay showed that four viral BTV proteins-VP3, VP4, VP5, and NS1-interacted with oISG15. We also found that the VP4 and NS1 proteins associated with ubiquitin via co-immunoprecipitation, and that oISG15 overexpression improved the stability of both proteins. Further results showed that the degradation of NS1 was involved in lysine 63-linked polyubiquitin. This suggested that oISG15 may interfere with NS1 degradation via the autophagy pathway. This study provides new insights on the interaction between BTV and ISG15, and enriches our understanding of the regulation and biological function of ISG15 with virus replication.

[Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model].

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-Erns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 106 TCID50 virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

[Using mouse model to evaluate the immune effect of DNA prime-protein boost strategies targeting Japanese encephalitis virus].

In order to evaluate the immune effect of the genotype Ⅰ Japanese encephalitis virus prM-E DNA vaccine and the prM-EⅢ fusion protein subunit vaccine on mice using DNA prime-protein boost strategy, the prM-E gene was inserted into the pVAX1 eukaryotic expression vector. The recombinant expression vector prM-E-pVAX1 was constructed as a DNA vaccine for initial immunity, and the recombinant prM-EⅢ fusion protein was obtained using a prokaryotic expression system as a subunit vaccine for enhanced immunity. Thirty two female BALB/c mice aged 4-6 weeks were randomly divided into four groups, and a prM-E-pVAX1 DNA vaccine group, a DNA prime-protein boost immune group, a prM-EⅢ subunit vaccine group, and a pVAX1 vector control group were set up. The specific antibody level in serum was monitored by ELISA, the neutralizing antibody titer was detected by plaque reduction neutralization, and the cellular immune responses induced by different vaccine immune groups were analyzed by cytokine expression abundance and lymphocyte proliferation experiments. The results showed that the neutralizing antibody titers induced by mice immunized with the DNA prime-protein boost strategy were close to that of the group immunized with the single prM-EⅢ subunit vaccine, but significantly higher than that of the group immunized with the single prM-E-pVAX1 DNA vaccine. DNA prime-protein boost strategies induced effective Th1/Th2 immune responses in mouse models, in particular the Th1 cell-mediated immune responses. This study provides a new immune strategy that may facilitate the prevention of Japanese encephalitis.

Development and validation of a DIVA ELISA for differentiating BEFV infected from vaccinated animals.

Inactivated vaccine is considered safe and used for prevention of bovine ephemeral fever in several endemic countries. To differentiate between BEFV-infected and vaccinated animals, we developed an ELISA capable of detecting infection-related antibodies against BEFV. Recombinant proteins, including N, P, M, L, GNS, α2, ß and γ, were expressed in E. coli and screened by Western blotting and ELISA. The results showed GNS, α2 and ß specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. A DIVA ELISA based on a C-terminal truncated form of GNS was developed, with 100% sensitivity and 98.0% specificity at a sample to positive-control optical density ratio (S/P) threshold of 0.18. Specificity analysis showed that the assay has no cross-reactivity with antisera of other common bovine viruses. Anti-GNS antibody appears at 3-4 days post infection (dpi) and persists up to 240-300 dpi in the experimentally infected cattle. Sero-epidemiological survey using sera collected from vaccinated cattle in an endemic area in Jiangsu Province revealed sero-positive rate of 2.36% (6/254), indicating that the DIVA ELISA could be used as a reliable diagnostic tool for differentiating BEFV infected from vaccinated animals.

ISG20 inhibits bluetongue virus replication.

ISG20 is an interferon-inducible exonuclease that inhibits virus replication. Although ISG20 is thought to degrade viral RNA, the antiviral mechanism and specificity of ISG20 remain unclear. In this study, the antiviral role of ovine ISG20 (oISG20) in bluetongue virus â€‹(BTV) infection was investigated. It was found that BTV infection up-regulated the transcription of ovine ISG20 (oISG20) in a time- and BTV multiplicity of infection (MOI)-dependent manner. Overexpression of oISG20 suppressed the production of BTV genome, proteins, and virus titer, whereas the knockdown of oISG20 increased viral replication. oISG20 was found to co-localize with BTV proteins VP4, VP5, VP6, and NS2, but only directly interacted with VP4. Exonuclease defective oISG20 significantly decreased the inhibitory effect on BTV replication. In addition, the interaction of mutant oISG20 and VP4 was weakened, suggesting that binding to VP4 was associated with the inhibition of BTV replication. The present data characterized the anti-BTV effect of oISG20, and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

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.