Abstract
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 A104R gene, which was replaced by the selection marker (GUS 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 GUS 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.
Highlights
African swine fever (ASF) is an infectious disease of suids, with lethalities up to 100%
In order to better characterize African swine fever virus (ASFV) biology and to identify viral antigens that could induce a protective immune response in the pig, different ASFV proteins putatively involved in viral replication and transcription, namely pA104R, have been studied in our lab. pA104R, known as histone-like protein, has an extensive sequence similarity to bacterial proteins that are implicated in genome replication and packaging [18,19]
Previous studies performed in our lab showed that pA104R, together with the ASFV-topoisomerase II, displays DNA supercoiling activity, suggesting that pA104R participates in the modulation of viral DNA topology and may be involved in viral DNA replication, transcription and packaging [22]
Summary
African swine fever (ASF) is an infectious disease of suids, with lethalities up to 100%. ASF is historically endemic in most of African Sub-Saharan countries and was first introduced into Europe via Portugal. Vaccines 2019, 7, 68 in 1957, afterwards spreading to neighboring countries, and to the Caribbean and Brazil. From 2007, the disease was introduced in Georgia, Armenia, Russia and Iran, and later spread into. In 2018, ASF was reported in Belgium and China, and during 2019 in Vietnam [5,6]. There is neither a vaccine nor a treatment against this global threat and the control of the disease relies on fast diagnosis, stamping out measures and trade bans of animals and pork products, giving rise to significant socio-economic losses in the affected areas [7,8]
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