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Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimer in Saccharomyces cerevisiae.

ABSTRACT: In an effort to control the outbreak of the African Swine Fever Virus (ASFV), there is an urgent need to develop an effective method to prevent the pandemic, including vaccines and diagnostic methods. The major capsid protein of ASFV p72 (B646L), which forms a trimer with each monomer adopting a double jelly roll fold, is the main component of the virus particle and major antigen of ASFV. Thus, the p72 protein may be considered an antigen candidate for vaccine and diagnostic development. However, the development of ASFV p72 trimer for the industry application, including veterinary usage, faces unavoidable challenges: firstly, the low cost of the antigen production is required in vaccine and diagnostic application; and, secondly, whether produced antigen folds in its native conformation. Here, based on the information provided by the atomic structure of p72, we have successfully performed rational mutagenesis on p72 trimers and expressed it in Saccharomyces cerevisiae with high yields. The cryo-EM structure of recombinant expressed p72 trimer is determined at 4.18 Å in resolution. The correlation coefficient between this structure and the ASFV virus structure is 0.77, suggesting a highly similar fold of this trimer with the native protein on the virus particle.

SUBMITTER: Meng K

PROVIDER: S-EPMC9239254 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: • Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. • Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.

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Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimer in Saccharomyces cerevisiae.

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