Abstract
BackgroundAfrican swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice.MethodsA homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model.ResultsThe PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well.ConclusionsThe PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.
Highlights
African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV)
Generation of the recombinant pseudorabies virus PRV‐ΔgE/ΔgI/ΔTK‐(CD2v) The recombinant pseudorabies virus PRV-ΔgE/ΔgI/ΔTK(CD2v) expressing ASFV CD2v protein was constructed by homologous recombination using the CRISPR/Cas9 technology
CD2v can be expressed in Human embryonic kidney (HEK) 293 T cells either by pcDNA3.1-EGFP-FlagCD2v-Flag transfection or PRV-ΔgE/ΔgI/ΔTK-(CD2v) infection
Summary
African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). No vaccine is commercially available for prevention, and the epidemic is still spreading. The virulent ASFV strains can cause acute hemorrhagic fever and lead to up to 100% mortality in domestic pigs and wild boar (Sus scrofa) [6, 12]. There is no commercial vaccine to prevent ASF, and the epidemic is still spreading. Various types of vaccines are under development to prevent ASF, including inactivated vaccines, live attenuated vaccines, subunit vaccines, DNA vaccines, virus vector vaccines, etc. The single or combination of ASFV CD2v, P30, P54, and other subunit vaccines show some protective effects [15, 16]. The "cocktail" immunization by using adenovirus vector vaccines carrying EP402R Δ PRR, p54, p72, and so on, and the recombinant B646l (p72), EP153R and EP402R (CD2v) vaccinia virus vaccines can all induce strong humoral and cellular immunity [17, 18]
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