Progress 03/01/22 to 02/28/25
Outputs
Target Audience:Researchers, regulators, animal health industries in the U.S. and internationally. Changes/Problems:During clinical sample validation, the anti-p30 and anti-CD2v mAb-based competitive ELISAs (cELISAs) exhibited suboptimal performance, specifically low sensitivity, limiting detection of antibody responses to 28 days post-vaccination/infection. To address this, we developed enhanced p30- and CD2v-based ELISA systems. These new systems demonstrate increased sensitivity, enabling detection of specific antibody responses at 14- and 21-days post-vaccination/infection, respectively. In the meantime, by utilizing mAbs targeting ASFV p30 and p72 proteins, we successfully developed two novel lateral flow assays for ASFV detection. p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources. What opportunities for training and professional development has the project provided?This project provided training and professional development of IBC, IACUC, working in BSL-3 facilities, pig handling, pig bleeding, clinical sample collection from pigs, ELISA, cell culture, and molecular biological techniques for our technicians and students. How have the results been disseminated to communities of interest?We presented our results of using the p30 based ELISA and CD2v based ELISA system to test the antibody responses of ASF live attenuated vaccine immunized pigs in three international meetings: 1) 2023 NAPRRS/NC229: International Conference of Swine Viral Diseases; Chicago, IL; 11/30-12/02/ 2023; Oral presentation "Development of live-attenuated vaccine to protect pigs against the contemporary pandemic African swine fever virus"; 2) 2023 International African Swine Fever Workshop; Beijing, China; 09/17-09/20/2023; Oral presentation "Cellular and humoral immune responses of ASF live attenuated vaccine candidate: KNB-LAV1"; 3) CRWAD 2024; Chicago, IL; 01/20-01/23/2024; Oral presentation "Live-attenuated vaccine against contemporary pandemic genotype II African swine fever virus". We introduced in detail about our ASFV variants quadplex real-time PCR assay in 2024 African Swine Fever Workshop for the Caribbean; Manhattan, KS; 06/24-06/28/2024. Our safe and efficacious cell-adapted ASF live attenuated vaccines and diagnostic tools were of interest to the audiences and have attracted widespread attention from international academic and industrial communities. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
• Collect field clinical samples from pigs naturally infected with ASFV in commercial swine farms in Vietnam and the Philippines. We collected clinical samples from pigs (n=166) naturally infected with ASFV across commercial swine farms. The samples represented a variety of biological matrices, including whole blood, serum, plasma, and dried blood on QIAcard FTA Elute Micro filter paper cards (Qiagen). All samples were tested positive for ASFV by ASFV-specific real-time PCR before being transferred to the United States. • Collect field clinical samples from ASF-free pigs in local farms in the United States. We collected clinical samples from ASF-free pigs (n=150) in local farms in the United States. The sample types included whole blood, serum, dried blood on QIAcard FTA Elute Micro filter paper cards (Qiagen), and ear punch biopsies. The samples were kept in our laboratory at Kansas State University. • Transfer the collected clinical samples from Vietnam and the Philippines to the United States. Samples that tested positive for ASFV using ASFV-specific real-time PCR were first submitted to the Plum Island Animal Disease Center (New York) for confirmatory testing. After receiving clearance from USDA APHIS, these samples were successfully transferred to our laboratory at the Biosecurity Research Institute, Kansas State University, KS. • Develop, optimize and validate cELISA system using p30 protein as the capture antigen and mAb3007 as the competitive antibody. We established themAb3007 based cELISA system. However, the performance ofthe cELISA system was not as good as we expected during validation with clinical samples(low sensitivity, can only detect theantibody responses 28 days post vaccination/infection). Thus, we developed a sensitivep30 based ELISA system to replacethe cELISA system. The p30 based ELISA system can detect the specific antibodyresponses 14 days post vaccination/infection. • Express CD2v protein with Baculovirus Expression System and generate anti-CD2v mAbs. We expressed and purified CD2v protein with Baculovirus Expression System and Plant Expression System. We immunized BALb/c mice with CD2v protein from both systems, and generated a panel of anti-CD2v mAbs (n=5). • Develop, optimize and validate cELISA system using CD2v protein as the capture antigen and anti-CD2v mAb as the competitive antibody. We established the anti-CD2v mAb based cELISA system. However, the performance ofthe cELISA system was not as good as we expected during validation with clinical samples(low sensitivity, can only detect theantibody responses 28 days post vaccination/infection). Thus, we developed a sensitiveCD2v based ELISA system to replacethe cELISA system. The CD2v based ELISA system can detect the specific antibodyresponses 21 days post vaccination/infection. • Develop, optimize and validate a reliable multiplex real-time PCR assay for ASFV variants testing. We developed a novel, sensitive, and reliable quadplex real-time PCR assay for detecting ASFV variants lacking key genes (I177L, EP402R, and MGF360-14L), either individually or in combination. The assay targets conserved regions within these genes, ensuring broad coverage of diverse ASFV genotypes. A porcine beta-actin (ACTB) internal control was incorporated to minimize false-negative results. Optimization and evaluation using spike-in tests demonstrated high sensitivity with a limit of detection (LOD) ranging from 1-10 plasmid copies or 0.1 TCID50 of ASFV isolates per reaction. No cross-reactivity was observed when testing serum samples from pigs infected with other common swine viruses. Further validation across a diverse panel of samples, comprising those from naturally ASFV-infected field pigs, experimentally ASFV-infected pigs, PBS-inoculated pigs, ASFV-free field pigs, and feral pigs, confirmed 100% specificity. This robust assay provides a valuable tool for rapid and accurate ASF surveillance and control efforts, facilitating the timely detection and mitigation of outbreaks caused by emerging ASFV variants.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Truong Q.L. , L. Wang , T.A. Nguyen, H.T. Nguyen, A.D. Le, G.V. Nguyen, A.T. Vu, P.T. Hoang, T.T. Le, H.T. Nguyen, H.T.T. Nguyen, H.T.L. Lai, D.A.T. Bui, L.M.T. Huynh, R. Madera, Y. Li, J. Retallick, F. Matias-Ferreyra, L.T. Nguyen, J. Shi. 2024. A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus. Viruses, 16 (8):1326.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wang L.*, J. Ren, J. Wang, H. Zhang J. Shi. 2024. Editorial: Advances and insights in the diagnosis of viral infections and vaccines development in animals. Front Microbiol, 15:1443858.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wang L.*, J. Kim, H. Kang, H. Park, M. Lee, S. Hong, C. Seo, R. Madera, Y. Li, A. Craig, J. Retallick, F. Matias-Ferreyra, E. Sohn, J. Shi. 2024. Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus. Front Microbiol, 15:1429808.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Wang, L.*, Y. Li, X. Zhang, R. Madera, H. Pantua, A. Craig, N. Muro, D. Li, J. Retallick, F.M. Ferreyra, Q.L. Truong, L.T. Nguyen, J. Shi. 2025. Specific Detection of African Swine Fever Virus Variants: A Novel Quadplex Real-Time PCR Assay with Internal Control. Microorganisms. Preprints 2025, 2025020813.
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Progress 03/01/23 to 02/29/24
Outputs
Target Audience:Researchers, regulators, animal health industries in the U.S. Changes/Problems: We established the mAb3007 based cELISA system. However, the performance of the cELISA system was not as good as we expected during validation with clinical samples (low sensitivity, can only detect theantibodies responses 28 days post vaccination/infection). Thus, we developed a sensitivep30 based ELISA system to replacethe cELISA system. The p30 based ELISA system can detect the specific antibodyresponses 14 days post vaccination/infection. Currently, we are working on validation (specificity) of the p30 based ELISA system with other common swine virus (CSFV, PRRSV, BVDV, PRV) infected serum samples. What opportunities for training and professional development has the project provided? This project provided training and professional development of IBC, IACUC, working in BSL-3 facilities, pig handling, pig bleeding, clinical sample collection from pigs, ELISA, cell culture, and molecular biological techniques for our technicians and students. How have the results been disseminated to communities of interest? We presented our results of using the p30 based ELISA and CD2v based ELISA system to test the antibody responses of ASF live attenuated vaccine immunized pigs in two international meetings: 1) 2023 NAPRRS/NC229: International Conference of Swine Viral Diseases; Chicago, IL; 11/30-12/02/ 2023; Oral presentation "Development of live-attenuated vaccine to protect pigs against the contemporary pandemic African swine fever virus"; 2) 2023 International African Swine Fever Workshop; Beijing, China; 09/17-09/20/2023; Oral presentation "Cellular and humoral immune responses of ASF live attenuated vaccine candidate: KNB-LAV1". What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to 1) Receive and put the clinical samples from pigs naturally infected with ASFVs into our long inventory system. 2) Validate the p30 based ELISA and CD2v based ELISA systems with more serum samples; 3) Optimize and validate multiplex real-time PCR assay for direct detection ASFV variants; 4) Share and disseminate research results through publications and presentations in domestic/international meetings and veterinary journals.
Impacts
What was accomplished under these goals?
1. Collect field clinical samples from pigs naturally infected with ASFV in commercial swine farms in Vietnam and the Philippines. • Obtained regulatory approvals for IBC and IACUC. • The reagents and materials for collecting field clinical samples have been sent to Vietnam and the Philippines. • Our collaborators have collected field clinical samples from pigs naturally infected with ASFV in local commercial swine farms. Challenges: ASF samples from Our collaborators (Vietnam and the Philippines) were delayed due to cross border transfer regulations. Clinical samples from pigs naturally infected with ASFVs was sent to Plum Island Animal Disease Center (New York) for testing in May, 2023. These samples are cleared for by USDA APHIS and are ready to be transferred to our laboratory at Kansas State University. Currently, we are waiting for the courier to transport the samples from Plum Island to Biosecurity Research Institute at Kansas State University, KS. 2. Collect field clinical samples from ASF-free pigs in local farms in the United States. • Obtained regulatory approvals for IBC and IACUC. • Collected 150 serum samples from 150 ASF-free pigs. • Collected 150 dry blood samples from 150 ASF-free pigs. • Collected 100 ear punch biopsy from 100 ASF-free pigs. 3. Transfer the collected clinical samples from Vietnam and the Philippines to the United States. ASF samples from Our collaborators (Vietnam and the Philippines) were delayed due to cross border transfer regulations. Clinical samples from pigs naturally infected with ASFVs was sent to Plum Island Animal Disease Center (New York) for testing in May, 2023. These samples are cleared for by USDA APHIS and are ready to be transferred to our laboratory at Kansas State University. Currently, we are waiting for the courier to transport the samples from Plum Island to Biosecurity Research Institute at Kansas State University, KS. 4. Develop, optimize and validate cELISA system using p30 protein as the capture antigen and mAb3007 as the competitive antibody. • Expressed and purified p30 protein with Baculovirus Expression System. • Cultured hybridomas which secreting mAb3007 and purified mAb3007 from the culture supernatant. • Horseradish peroxidase (HRP) labeled mAb3007. •Established the HRP-mAb3007 based cELISA system. However, the performance of HRP-mAb3007 based cELISA system was not as good as we expected (low sensitivity, can only detect theantibodies responses 28 days post vaccination/infection). Thus, we developed a sensitive p30 based ELISA to replacethe cELISA. The p30 based ELISA can detect the specific antibodyresponses 14 days post vaccination/infection. Currently, we are working on validation (specificity) of the p30 based normal ELISA with other common swine virus (CSFV, PRRSV, BVDV, PRV) infected serum samples. 5. Express CD2v protein, generate anti-CD2v mAbs, establish and validate the anti-CD2v mAb based cELISA system • Expressed and purified CD2v protein with Baculovirus Expression System. • Expressed and purified CD2v protein with Plant Expression System. • Immunized BALb/c mice with CD2v protein from both systems, and generated a panel of anti-CD2v mAbs (n=5). •Established the anti-CD2v mAb based cELISA system. 6. Develop, optimize and validate a reliable multiplex real-time PCR assay for ASFV variants testing. • Established sequence databases of I177L gene, CD2v gene and MGF gene which collected all available ASFV sequences from GenBank, ASFV sequences in our lab and other published sequences. • Completed sequence alignments, primer and probe design. • Prepared the standard template plasmids: pUC57-I177L, pUC57-CD2v and pUC57-MGF. • Orderedand received the commercially available multiplex reagents: iQ Multiplex Powermix from Bio-Rad; QIAGEN Multiplex PCR Kit from Qiagen; Platinum™ Multiplex PCR Master Mix from ThermoFisher, Multiplex PCR 5X Master Mix from New England Biolabs, andPath-ID Multiplex One-Step RTPCR Kit (Applied Biosystems/Life Technologies). Next step: Compare the performance of different multiplex reagents with designed primers and probes and optimize the ASFV variants multiplex real-time PCR system. Validate (diagnostic specificity and sensitivity) the ASFV variants multiplex real-time PCR system with clinical samples.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Wang L., R. Madera, Y. Li, D.P. Gladue, M.V. Borca, M.T. McIntosh, J. Shi. 2023. Development of porcine monoclonal antibodies with in vitro neutralizing activity against Classical Swine Fever virus from C-Strain E2-specific single B cells. Viruses, 15(4):863.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Truong Q.L., L. Wang, T.A. Nguyen, T.H. Nguyen, S.D. Tran, A.T. Vu, A.D. Le, V.G. Nguyen, P.T. Hoang, Y.T. Nguyen, T.L. Le, T.N. Van, T. Huynh, H. Lai, R. Madera, Y. Li, J. Shi, L.T. Nguyen. 2023. A cell-adapted live-attenuated vaccine candidate protects pigs against the homologous strain VNUA-ASFV-05L1, a representative strain of the contemporary pandemic African Swine Fever virus. Viruses, 15(10):2089.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wang L., S. Mi, R. Madera, Y. Li, W. Gong, C. Tu, J. Shi. 2022. A novel competitive ELISA for specifically measuring and differentiating immune responses to Classical Swine Fever C-strain vaccine in pigs. Viruses, 14:1544.
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Progress 03/01/22 to 02/28/23
Outputs
Target Audience:Researchers, regulators and animal health industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training and professional development of IBC, IACUC, working inBSL-3 facilities, pig handling, pig bleeding, clinical sample collection from pigs, ELISA, cell culture, and molecular biological techniques for our technicians andstudents. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to 1) Continue to work with our collaborators to collect and transfer clinical samples from foreign countries to U.S.; 2) establish and validate thecELISA systems; 3) establish and validate multiplex real-time PCR assay for direct detection ASFV variants;4)Share and disseminate research results throughpublications and presentations in domestic/international meetings and veterinary journals.
Impacts
What was accomplished under these goals?
•Collect field clinical samples from pigs naturally infected with ASFV in commercial swine farms in Vietnam and the Philippines. 1) Obtained regulatory approvals for IBC and IACUC. 2) The reagents and materials for collecting field clinical samples have been sent to Vietnam and the Philippines. 3) Our collaborators have started to collect field clinical samples from pigs naturally infected with ASFV in local commercial swine farms. •Collect field clinical samples from ASF-free pigs in local farms in the United States. 1) Obtained regulatory approvals for IBC and IACUC. 2) Collected 150 serum samples from 150 ASF-free pigs. 3) Collected 150 dry blood samples from 150 ASF-free pigs. 4) Collected 100 ear punch biopsy from 100 ASF-free pigs. •Transfer the collected clinical samples from Vietnam and the Philippines to the United States. No data to report yet. •Develop, optimize and validate cELISA system using p30 protein as the capture antigen and mAb3007 as the competitive antibody. 1)Expressed and purified p30 protein with Baculovirus Expression System. 2) Cultured hybridomas which secreting mAb3007 and purified mAb3007 from the culture supernatant. 3) Horseradish peroxidase (HRP) labeled mAb3007. •Express CD2v protein with Baculovirus Expression System and generate anti-CD2v mAbs. 1) Expressed and purified CD2v protein with Baculovirus Expression System. 2) Expressed and purified CD2v protein with Plant Expression System. 3) Immunized BALb/c mice with CD2v protein from both systems to generate anti-CD2v mAbs. •Develop, optimize and validate cELISA system using CD2v protein as the capture antigen and anti-CD2v mAb as the competitive antibody. 1) Established CD2v based ELISA system to test anti-CD2v antibodies in pig serum.This CD2v based ELISA system can test the antibodies in pig serum 21 days post of immunization with subunit vaccine.Currently, working on validate the CD2v based ELISA system with more serum samples. • Develop, optimize and validate a reliable multiplex real-time PCR assay for ASFV variants testing. 1) Established sequence databases of I177L gene, CD2v gene and MGF gene which includeall available ASFV sequences from GenBank, ASFV sequences in our lab and other published sequences.Currently working on sequence alignment, primer and probe design.
Publications
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