A blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for detecting ASFV antibodies.

African swine fever virus (ASFV) is a highly lethal pathogen of domestic and wild pigs. Due to no vaccines or drugs available, early accurate diagnosis and eradication of infected animals are the most important measures for ASFV prevention and control. Bluetongue virus (BTV) core-like particles (CLPs) are non-infectious hollow nanoparticles assembled from the BTV VP3 and VP7 proteins, which could be used as a platform for presenting foreign epitopes. In this study, the secondary structure of BTV VP7 protein was analyzed and predicted using the IEDB Analysis resource. Based on the prediction results of the VP7 protein, the chimeric CLPs with an ASFV P54 epitope were successfully prepared through the BAC-to-BAC baculovirus expression system and sucrose gradient centrifugation. Based on the chimeric CLPs and mAb 2E4 against AFSV P54 epitope, a blocking ELISA for detecting AFSV antibodies was established, and its reaction conditions were optimized. Through comprehensive evaluation of the method, the results showed the chimeric CLPs-based blocking ELISA displayed the best detection performance, with an AUC of 0.9961, a sensitivity of 97.65%, and a specificity of 95.24% in ROC analysis. Compared with western blot and a commercial c-ELISA for detecting anti-ASFV antibodies, this method had an excellent agreement of 96.35% (kappa value = 0.911) and 97.76% (kappa value = 0.946) with the other tests, respectively. This ELISA also had high repeatability, with CV < 10%, and no cross-reaction with the serum antibodies against other swine viruses or Orbivirus. In brief, this was the first report on developing a blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for serological diagnosis of ASFV.

Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus.

African swine fever (ASF) is an infectious disease of domestic and wild pigs, caused by ASF virus (ASFV). In this study, a triplex real-time PCR assay was developed to detect and differentiate the gene-deleted and wild-type ASFV strains. Three pairs of primers and probes were designed to target the conserved region of B646L gene (p72), MGF_360-14L gene (located in the middle of MGF360-505R gene) and CD2v gene, respectively. Gene-deleted (with MGF360-505R and / or CD2v genes deletion) and wild-type ASFV strains were detected specifically and simultaneously by the assay developed without cross-reactions with other nucleic acids of PCV-2, CSFV, PRRSV, FMDV or SVA. The detection limits of the triplex rPCR were 7.9 copies, 9.7 copies, and 9.6 copies of standard plasmid DNA containing B646L gene, MGF_360-14L gene and CD2v gene, respectively. A total of 1215 field samples were tested in parallel by the triplex rPCR and real-time PCR recommended by OIE, and the B646L gene detection results were completely consistent between these two assays. The triplex rPCR assay was successfully developed to identify pigs infected with wild-type ASFV strains or immunized with the ASFV gene-deleted vaccine.

Detection of a novel highly pathogenic H7 influenza virus by duplex real-time reverse transcription polymerase chain reaction.

On February 19, 2017, China announced that the mutant H7N9 virus appeared in human cases, which showed molecular characteristic of highly pathogenic virus for poultry. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for distinguish between highly pathogenic H7 virus and low pathogenic H7 virus. The sensitivity, specificity, stability and conformance tests were conducted for this method. The data showed that the new method is sensitive. The minimum detection limit for the RNA of highly pathogenic H7 virus is 0.0052fg and the minimum detection limit for the RNA of low pathogenic H7 virus is 0.36fg. The method gave specific results in detecting novel highly pathogenic H7 virus and will play an important role in the rapid identification of novel highly pathogenic H7 virus.

A new immunoassay of serum antibodies against Peste des petits ruminants virus using quantum dots and a lateral-flow test strip.

A fast and ultrasensitive test-strip system combining quantum dots (QDs) with a lateral-flow immunoassay strip (LFIAS) was established for detection of Peste des petits ruminants virus (PPRV) antibody. The highly luminescent water-soluble carboxyl-functionalized QDs were used as the signal output and were conjugated to streptococcal protein G (SPG), which was capable of binding to immunoglobulin G (IgG) from many species through an amide bond to capture the target PPRV IgGs. The PPRV N protein, which was immobilized on the detection zone of the test strip, was expressed by transfecting recombinant Bacmid-PPRV-N with Lipofect into Sf9 insect cells. When exposed to PPRV IgG, QD-SPG bound to PPRV N protein, resulting in the formation of a complex that subsequently produced a bright fluorescent band in response to 365 nm ultraviolet excitation. Sensitivity evaluation showed that the QD-LFIAS limit of detection (LOD) for PPRV antibody was superior to competitive enzyme-linked immunosorbent assay (c-ELISA) and the immunochromatographic strip. No cross reaction was observed when the positive sera of bluetongue virus, canine distemper virus, goat pox virus, and foot-and-mouth disease virus were tested. Further evaluation using field samples indicated that the diagnostic specificity and sensitivity of the QD-LFIAS was 99.47 and 97.67 %, respectively, with excellent agreement between QD-LFIAS and c-ELISA. The simple analysis step and objective results that can be obtained within 15 min indicate that this new method shows great promise for rapid, sensitive detection of PPRV IgG for onsite, point-of-care diagnosis and post vaccination evaluation (PVE). Graphical Abstract Ultrasensitive fluorescent QD immunochromotography in combination with recombinant PPRV N protein could be used to detect PPRV antibody in serum.

[Cloning and expression of the prokaryotic expression vectors of phytoplasma immunodominant membrane protein A and preparation of its antiserum].

OBJECTIVE: To construct the prokaryotic expression vector of phytoplasma immunodominant membrane protein A (IdpA) in prokaryotic cell, express and purify the IdpA and prepare its antiserum. METHODS: With the recombinant plasmid pMD18-T-IdpA as templates, IdpA gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a(+) by endonuclease reaction and T4 DNA ligase reaction. Then the recombinant plasmid pET-28a(+)-IdpA was transformed into E.coli BL21 (DE3). After confirmed by PCR and double enzyme digestion, the recombinant protein IdpA was expressed under IPTG induction and purified. The purified product was used to immunize BALB/c mice to prepare its antiserum. IdpA-specific mouse antiserum was identified by ELISA and Westerrn blotting. RESULTS: The prokaryotic vectors of pET-28a(+)-IdpA were constructed successfully and the recombinant protein IdpA was induced to express stably in the E.coli BL21. The purity of IdpA was up to over 90%. In the BALB/c mice immunized by the purified IdpA, the titre of IdpA-specific antiserum was as high as 1:320 000. CONCLUSION: The recombinant protein IdpA was expressed successfully in E.coli and the IdpA-specific antiserum was prepared.