Diagnostic assay and vaccine vector development for African Swine Fever Virus

Yuan, Fangfeng

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https://hdl.handle.net/2142/117640 Copy

Description

Title

Diagnostic assay and vaccine vector development for African Swine Fever Virus

Author(s)

Yuan, Fangfeng

Issue Date

2022-10-25

Director of Research (if dissertation) or Advisor (if thesis)

Fang, Ying

Doctoral Committee Chair(s)

Fang, Ying

Committee Member(s)

• Lau, Gee
• Wang, Leyi
• Dilger, Ryan N.
• Jarosinski, Keith W.

Department of Study

Pathobiology

Discipline

VMS - Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• ASFV
• mAb
• bELISA
• diagnostic
• PRV1
• vector
• vaccine

Abstract

African swine fever (ASF) is a highly contagious swine viral disease featured by high morbidity and mortality in domestic swine populations. The introduction of ASF to China and other Asian countries resulted in devastating impacts on the countries’ economy. Since no vaccines are available yet in the market, the only strategy to control ASF is through quick identification of infected animals and subsequent implementation of strict biosecurity measures. Thus, highly sensitive and specific diagnostic reagents and assays are urgently needed, as well as novel strategies for ASF vaccine development. In the present study, we generated a panel of specific monoclonal antibodies (mAbs) against selected immunogenic ASFV proteins, including p10, p14.5, p22, p49, p54, p72, and CD2v. These proteins were selected based on their key functions in viral replication and immunogenicity. These mAbs were initially screened by immunofluorescent assay using in vitro expression system. The antibody reactivity was confirmed in virus-infected cells. Their application in the detection of ASFV infection was further tested using the methods of Western blotting, immunoprecipitation, and ELISA. The availability of these mAbs provides an important tool in aid of ASFV diagnostics and research. We further developed a highly specific and repeatable blocking ELISA (bELISA) using a recombinant p30 protein as the antigen combined with biotinylated mAb against p30 as the detection antibody. Receiver operating characteristic (ROC) analysis of the data calculated an optimal percentage of inhibition (PI) cutoff value of 45.92%, giving a diagnostic sensitivity of 98.11% and diagnostic specificity of 99.42%. The coefficient of variation of an internal quality control serum was 6.81% for between runs, 6.71% for within run, and 6.14% for within plate. A time course study of infected pigs showed that bELISA was able to detect seroconversion as early as 7 days post-inoculation. Evaluation on an alphavirus-expressing p30 vaccination study indicated that the assay was able to detect p30 antibody response in oral fluids at 19 days post-vaccination (dpv) and then throughout the study. These results demonstrate that bELISA can be used as an alternative serological test for detecting ASFV infection in aid of disease surveillance. In our previous study, Porcine respirovirus 1 (PRV1) KS 17-258 strain was isolated and characterized molecularly. Members in paramyxoviruses have the potential to serve as a viral vector for vaccine development. In this study, we stablished a reverse genetic system of the PRV1 KS17-258 strain, which was further explored as a viral vector to express ASFV p30 and p54 proteins. Recombinant viruses expressing p30/p54 exhibited similar growth kinetics to cloned and parental viruses. The expression cassettes of p30 and p54 genes in the recombinant viruses remained stable for more than 10 passages in cell culture, and the stability was further confirmed in nursery pigs inoculated with those recombinant viruses. Viral RNA containing intact p30 or p54 gene was detected in multiple tissues, including nasal turbinate, trachea, and lung at 4 days post-inoculation (dpi), while viral RNA in nasal turbinate can be detected throughout the 28 dpi. Of note, infected pigs generated significant antibody responses against p30 and p54 proteins, which confirmed that these ASFV antigens were expressed by the recombinant viruses in infected pigs. This study establishes a novel platform for future development of vectored vaccines against swine diseases. In all, the diagnostic reagents (mAb), assay (bELISA), and vaccine vector platform (PRV1 vector) developed in this dissertation serve as important preparedness for ASF control.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Fangfeng Yuan

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