Classical swine fever (CSF) is caused by classical swine fever virus (CSFV) and has led to huge ecnomic losses for the pig industry worldwide. Although vaccination and other control measures have been carried out, it is essential to establish a rapid and valid method for CSF vaccination monitoring and clinical diagnosis. CSFV E2 protein has been well-known as a major antigen for antibody detection. It is significant to improve affinity between E2 protein and CSFV antibody for a better performance of detection method. In this study, a recombinant E2 extracellular protein (aa 1-331), which has a native homodimer conformation and has a high affinity with anti-CSFV-E2 monoclonal antibody WH303, was expressed using Bac-to-Bac baculovirus expression system. A novel immunochromatographic test strip based on the recombinant CSFV E2 protein was developed for CSFV antibody detection. The sensitivity of this strip for detecting CSFV standard positive serum was 1:102400, 4 times higher than that of the previously developed CnC2 test strip. No cross reaction with antibodies of other swine viruses was observed. The detection of clinical swine serum samples (n=138) demonstrated that the agreements of this E2 test strip with three commercial ELISA kits were 88.40% (122/138), 86.23% (119/138), and 96.38% (133/138), respectively. Our data indicated that a novel E2 test strip with higher sensitivity has been developed and can be applied for clinical sample detections, providing a new powerful and simple approach for CSFV antibody monitoring.

Influenza A virus (IAV), a deadly zoonotic pathogen, occasionally cross-species transmission among humans, swine and avian. The ectodomain of matrix protein 2 (M2e) is highly conserved in IAV, and multi-copy M2e from different species are usually displayed on the surface of nanoparticles to improve immunogenicity and constitute universal IAV nanovaccines. In our previous study, three M2e were inserted into the C-terminal of Cap protein of porcine circovirus type 2 (PCV2) to form a universal nanovaccine that protects PCV2 and different subtypes of IAV. Howerer, M2e adopts at least two converted conformations, and the intermolecular linker of M2e enhances the conformational instability, which limits the recognition of B cell receptors and production of high-level antibodies. Here, we report that the permutation of the M2e affects immune effect of nanovaccines. Three M2e derived from humans, swine and avian IAV were inserted into the C-terminal of the Cap protein to form nanovaccines. Immunoprotective effects of different M2e arrangements were explored in mice. Results showed that the M2e closest to the surface of nanoparticle induced the most efficient protection against IAV derived from corresponding species. The results will help to develop more effective universal IAV and PCV2 bivalent nano-vaccines, as well as universal IAV vaccines for specific species.

Influenza A virus (IAV), a deadly zoonotic pathogen, occasionally cross-species transmission among humans, swine and avian. The ectodomain of matrix protein 2 (M2e) is highly conserved in IAV and has been widely concerned in the development of universal influenza vaccines. Due to low immunogenicity, multi-copy M2e are usually displayed on the surface of nanoparticles to constitute universal nanovaccines. Here, we report that the permutation of the M2e affects the immune effect of the nanovaccine. Three M2e derived from humans, swine and avian IAV were inserted into the C-terminal of the Cap protein of porcine circovirus type 2 (PCV2) to form self-assembled nanovaccine. Immunoprotective effects of different M2e arrangements were explored in mice. Results showed that the M2e closest to the surface of nanoparticle induced the most efficient protection against IAV derived from corresponding species. The results will help in the development of more effective universal influenza vaccines, especially for specific species.

Bluetongue (BT) is a non-contact infectious disease of domestic or wild ruminants caused by the Bluetongue virus (BTV). It is transmitted by Culicoides biting midges. BTV mainly infect sheep, and animals infected with BTV usually show clinical symptoms such as fever, mucosal edema, and ulcers. BTV virus is an icosahedral symmetric RNA virus with 27 different serotypes. BTV consists of 7 structural proteins (VP1-VP7) and 4 non-structural proteins (NS1, NS2, NS3/NS3a, NS4, NS5). The VP3 encoded by the L3 gene is relatively high conserved structural protein, and constitute the inner symmetric icosahedral core shell of virus particle. In this study, VP3 recombinant protein of bluetongue I virus was successfully expressed and purified by prokaryotic expression system. Moreover, to increase the amount of soluble protein, we used chaperone protein ptf16 and two fusion proteins NusA and TRX. The results show that chaperone protein can increase the solubility of VP3. Then the expression conditions were optimized and VP3 was purified by nickel affinity chromatography. The VP3 immunize Balb/c mice and the results show that the serum titer is 1:1.28×105. In the Dot-ELISA assay, we found recombinant protein VP3 react with the serum from immunized mice by inactivate BTV-1. The results of IFA showed that the antibody produced by immunized mice with recombinant protein VP3 could react with the VP3 protein expressed in 293T cell. In conclusion, we expressed the recombinant protein VP3 with the same conformation as eukaryotic expression system and had same immunogenicity with inactivated virus, which laid a foundation for further study on the structure and function of BTV.