J Vet Sci.  2017 Aug;18(S1):361-370. 10.4142/jvs.2017.18.S1.361.

Generation, characterization, and application in serodiagnosis of recombinant swine vesicular disease virus-like particles

• Xu W
• Goolia M
• Salo T
• Zhang Z
• Yang M

• KMID: 2412561
• DOI: http://doi.org/10.4142/jvs.2017.18.S1.361

Abstract

Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.

Keyword

enzyme-linked immunosorbent assay; serological tests; swine vesicular disease virus; virus-like particles

MeSH Terms

Animals
Blotting, Western/veterinary
*Enterovirus B, Human
Enzyme-Linked Immunosorbent Assay/veterinary
Fluorescent Antibody Technique/veterinary
Microscopy, Electron/veterinary
Serologic Tests/methods/veterinary
Swine
Swine Vesicular Disease/*diagnosis/virology

Figure

• 

  Fig. 1 Swine vesicular disease (SVD) virus-like particles (VLP) detection using fluorescence (A) and electron microscopy (B). (A) Sf9 cells were grown on glass slides, infected with recombinant baculovirus at multiplicities of infection 0.1, and harvested at 24 h post-infection. SVD-VLP were stained with the Mab F44-SVD and then with Alexa Fluor 594 (red; Invitrogen). (B) Sf9 cells were infected with recombinant baculovirus and then lysed. SVD-VLP were purified, negatively stained, and examined via transmission electron microscopy. Scale bar = 100 nm.

• 

  Fig. 2 Western blot analysis of swine vesicular disease virus (SVDV) and swine vesicular disease (SVD) virus-like particles (VLP) capsid proteins. The mock-infected Sf9 cell lysates (lane 1), purified SVDV (lane 2), and SVD-VLP (lane 3) were separated in SDS-PAGE and transferred to nitrocellulose membranes. The SVD capsid proteins were probed with an anti-mouse SVD serum and visualized with horseradish peroxidase-conjugated goat anti-mouse antibody and then 3, 3′-diaminobenzidine.

• 

  Fig. 3 Liquid chromatography tandem mass spectrometry identification of swine vesicular disease virus-like particles capsid proteins. The protein bands corresponding to positions indicated in Fig. 2 were separately excised from SDS-PAGE, destained, processed with trypsin, and identified by mass spectrometry peptide fingerprinting as the VP0, VP1, and VP3 proteins. Fig. 3 shows the amino acid sequence of the SVD virus capsid polyprotein and indicates the 9 unique tryptic peptides identified within the protein sequence. Cleavage sites are shown by arrows.

• 

  Fig. 4 Comparison of swine vesicular disease virus (SVDV) antigen (Ag) competitive enzyme-linked immunosorbent assay (cELISA), swine vesicular disease (SVD) virus-like particles (VLP) cELISA, and virus neutralization test (VNT) results for SVD-specific antibody detection. The sera were collected at different times in the infection course of ten pigs experimentally inoculated with SVDV. (A) Results are expressed as means of percent inhibitions ± SD from ten pigs for cELISA. Samples at 5, 7, 9, and 28 dpi from six pigs were analyzed using the VNT. (B) Scatter plot representing the percentage inhibition in SVDV-Ag cELISA vs. SVD-VLP cELISA for 120 serial blood samples from ten pigs. The line is fitted by linear regression (r2 = 0.96, p < 0.0001). (C) Scatter plot representing the percentage inhibition in SVD-VLP cELISA vs. VNT titers for 24 serial blood samples from six pigs. The line is fitted by linear regression (r2 = 0.64, p < 0.0001).

• 

  Fig. 5 Swine vesicular disease (SVD) immunoglobulin (Ig) M-specific antibody detection using swine vesicular disease virus (SVDV) antigen (Ag) and SVD virus-like particles (VLP) in IgM-specific enzyme-linked immunosorbent assay (ELISA). The sera were sequentially collected from ten pigs experimentally inoculated with SVDV. (A) Results are expressed as means of P/N absorbance ratio ± SD from 10 pigs. (B) Scatter plot representing the P/N ratio in SVDV-Ag-IgM ELISA vs. SVD-VLP-IgM ELISA for 120 serial blood samples from ten pigs. The line is fitted by linear regression (r2 = 0.87, p < 0.0001).

• 

  Fig. 6 Swine vesicular disease (SVD) immunoglobulin (Ig) G-specific antibody detection using swine vesicular disease virus (SVDV) antigen (Ag) and SVD virus-like particles (VLP) in IgG-specific enzyme-linked immunosorbent assay (ELISA). The sera were sequentially collected from 10 pigs experimentally inoculated with SVDV. (A) Results are expressed as means of P/N absorbance ratio ± SD from ten pigs. (B) Scatter plot representing the P/N ratio in SVDV-Ag-IgG ELISA vs. SVD-VLP-IgG ELISA for 120 serial blood samples from ten pigs. The line is fitted by linear regression (r2 = 0.72, p < 0.0001).

Reference

1. Basavappa R, Syed R, Flore O, Icenogle JP, Filman DJ, Hogle JM. Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 Å resolution. Protein Sci. 1994; 3:1651–1669.

2. Brocchi E, Berlinzani A, Gamba D, De Simone F. Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus. J Virol Methods. 1995; 52:155–167.

3. De Clercq K. Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA. J Virol Methods. 1998; 70:7–18.

4. Dekker A, van Hemert-Kluitenberg F, Baars C, Terpstra C. Isotype specific ELISAs to detect antibodies against swine vesicular disease virus and their use in epidemiology. Epidemiol Infect. 2002; 128:277–284.

5. Hammond GW, Hazelton PR, Chuang I, Klisko B. Improved detection of viruses by electron microscopy after direct ultracentrifuge preparation of specimens. J Clin Microbiol. 1981; 14:210–221.

6. Heckert RA, Brocchi E, Berlinzani A, Mackay DK. An international comparative analysis of a competitive ELISA for the detection of antibodies to swine vesicular disease virus. J Vet Diagn Invest. 1998; 10:295–297.

7. Inoue T, Suzuki T, Sekiguchi K. The complete nucleotide sequence of swine vesicular disease virus. J Gen Virol. 1989; 70:919–934.

8. Inoue T, Yamaguchi S, Kanno T, Sugita S, Saeki T. The complete nucleotide sequence of a pathogenic swine vesicular disease virus isolated in Japan (J1'73) and phylogenetic analysis. Nucleic Acids Res. 1993; 21:3896.

9. Jore J, De Geus B, Jackson RJ, Pouwels PH, Enger-Valk BE. Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. J Gen Virol. 1988; 69:1627–1636.

10. Karakas M, Palfi V. [Diagnostic difficulties during the serological examination of swine vesicular disease]. Magy Allatorvosok Lapja. 1996; 51:595–597. Hungarian.

11. Ko YJ, Choi KS, Nah JJ, Paton DJ, Oem JK, Wilsden G, Kang SY, Jo NI, Lee JH, Kim JH, Lee HW, Park JM. Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 2005; 12:922–929.

12. Lin F, Kitching RP. Swine vesicular disease: an overview. Vet J. 2000; 160:192–201.

13. Nijhar SK, Mackay DK, Brocchi E, Ferris NP, Kitching RP, Knowles NJ. Identification of neutralizing epitopes on a European strain of swine vesicular disease virus. J Gen Virol. 1999; 80:277–282.

14. Racaniello VR. Picornaviridae: the viruses and their replication. In : Knipe DM, Howley PM, editors. Fields Virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins;2007. p. 795–838.

15. Seechurn P, Knowles NJ, McCauley JW. The complete nucleotide sequence of a pathogenic swine vesicular disease virus. Virus Res. 1990; 16:255–274.

16. Vratskikh O, Stiasny K, Zlatkovic J, Tsouchnikas G, Jarmer J, Karrer U, Roggendorf M, Roggendorf H, Allwinn R, Heinz FX. Dissection of antibody specificities induced by yellow fever vaccination. PLoS Pathog. 2013; 9:e1003458.

17. World Organisation for Animal Health (OIE). Swine vesicular disease. OIE Biological Standards Commission. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Volume 2:6th ed. Paris: Office International des Epizooties;2008. p. 1139–1145.

18. Yang M, Clavijo A. Monoclonal antibody against swine vesicular disease virus. Hybridoma. 2008; 27:325.

19. Yang M, Clavijo A, Suarez-Banmann R, Avalo R. Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus. Vet Immunol Immunopathol. 2007; 115:126–134.

20. Ypma-Wong MF, Dewalt PG, Johnson VH, Lamb JG, Semler BL. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the p1 capsid precursor. Virology. 1988; 166:265–270.