Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate
African swine fever (ASF) is a fatal viral disease of domestic swine and wild boar, considered one of the main threats for global pig husbandry. Despite enormous efforts, to date, neither the classical vaccine formulations nor the use of protein subunits proved to be efficient to prevent this diseas...
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| Format: | Article |
| Language: | English |
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MDPI AG
2019-07-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/7/3/68 |
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| author | Ferdinando B. Freitas Margarida Simões Gonçalo Frouco Carlos Martins Fernando Ferreira |
| author_facet | Ferdinando B. Freitas Margarida Simões Gonçalo Frouco Carlos Martins Fernando Ferreira |
| author_sort | Ferdinando B. Freitas |
| collection | DOAJ |
| description | African swine fever (ASF) is a fatal viral disease of domestic swine and wild boar, considered one of the main threats for global pig husbandry. Despite enormous efforts, to date, neither the classical vaccine formulations nor the use of protein subunits proved to be efficient to prevent this disease. Under this scenario, new strategies have been proposed including the development of disabled infectious single cycle (DISC) or replication-defective mutants as potential immunizing agents against the ASF virus (ASFV). In this study, we describe the methodology to generate an ASFV-DISC mutant by homologous recombination, lacking the <i>A104R</i> gene, which was replaced by the selection marker (<i>GUS</i> gene). The recombinant viruses were identified when the infected cells acquired a blue color in the presence of X-Gluc (100 µg/mL), which is the substrate for the <i>GUS</i> gene. Since these viral particles result from loss-of-function mutations, being unable to replicate, helper-cell lines expressing the viral pA104R protein were produced. Vero and COS-1 cell lines were transfected by different methods, both physical and chemical, in order to stably express the ASFV-pA104R. Best results were obtained by using Lipofectamine 2000 and Nucleofection methodology of Vero with the pIRESneo vector and by using Flp-FRT site-directed recombination technology system in Flp-In CV-1 cells (transformed COS-1 cells with a single integration site in a transcriptional active region). In order to ensure an efficient and stable integration of the viral ORF on the host cellular genome, the maintenance of the insert was verified by PCR and its expression by immunofluorescence and immunoblot analysis. Although the isolation of the recombinant virus was not achieved, the confirmation of ASFV-ΔA104R sequence, and the detection of the recombinant mutant through three passages, suggest that this approach is feasible and could be a potential strategy to generate safe and efficient DISC vaccine candidates. |
| first_indexed | 2024-04-11T22:29:00Z |
| format | Article |
| id | doaj.art-0b3f57bf77524bafb0255d018e217200 |
| institution | Directory Open Access Journal |
| issn | 2076-393X |
| language | English |
| last_indexed | 2024-04-11T22:29:00Z |
| publishDate | 2019-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj.art-0b3f57bf77524bafb0255d018e2172002022-12-22T03:59:34ZengMDPI AGVaccines2076-393X2019-07-01736810.3390/vaccines7030068vaccines7030068Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine CandidateFerdinando B. Freitas0Margarida Simões1Gonçalo Frouco2Carlos Martins3Fernando Ferreira4CIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, PortugalCIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, PortugalCIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, PortugalCIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, PortugalCIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, PortugalAfrican swine fever (ASF) is a fatal viral disease of domestic swine and wild boar, considered one of the main threats for global pig husbandry. Despite enormous efforts, to date, neither the classical vaccine formulations nor the use of protein subunits proved to be efficient to prevent this disease. Under this scenario, new strategies have been proposed including the development of disabled infectious single cycle (DISC) or replication-defective mutants as potential immunizing agents against the ASF virus (ASFV). In this study, we describe the methodology to generate an ASFV-DISC mutant by homologous recombination, lacking the <i>A104R</i> gene, which was replaced by the selection marker (<i>GUS</i> gene). The recombinant viruses were identified when the infected cells acquired a blue color in the presence of X-Gluc (100 µg/mL), which is the substrate for the <i>GUS</i> gene. Since these viral particles result from loss-of-function mutations, being unable to replicate, helper-cell lines expressing the viral pA104R protein were produced. Vero and COS-1 cell lines were transfected by different methods, both physical and chemical, in order to stably express the ASFV-pA104R. Best results were obtained by using Lipofectamine 2000 and Nucleofection methodology of Vero with the pIRESneo vector and by using Flp-FRT site-directed recombination technology system in Flp-In CV-1 cells (transformed COS-1 cells with a single integration site in a transcriptional active region). In order to ensure an efficient and stable integration of the viral ORF on the host cellular genome, the maintenance of the insert was verified by PCR and its expression by immunofluorescence and immunoblot analysis. Although the isolation of the recombinant virus was not achieved, the confirmation of ASFV-ΔA104R sequence, and the detection of the recombinant mutant through three passages, suggest that this approach is feasible and could be a potential strategy to generate safe and efficient DISC vaccine candidates.https://www.mdpi.com/2076-393X/7/3/68ASFVpA104Rhelper cell lineDISCvaccine |
| spellingShingle | Ferdinando B. Freitas Margarida Simões Gonçalo Frouco Carlos Martins Fernando Ferreira Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate Vaccines ASFV pA104R helper cell line DISC vaccine |
| title | Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate |
| title_full | Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate |
| title_fullStr | Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate |
| title_full_unstemmed | Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate |
| title_short | Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line—A Potential Methodology for the Production of a Vaccine Candidate |
| title_sort | towards the generation of an asfv pa104r disc mutant and a complementary cell line a potential methodology for the production of a vaccine candidate |
| topic | ASFV pA104R helper cell line DISC vaccine |
| url | https://www.mdpi.com/2076-393X/7/3/68 |
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