RESUMO
In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.
Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/imunologia , Antígenos Virais/imunologia , Vacinas Virais/imunologia , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Alphavirus/genética , Alphavirus/metabolismo , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Imunização Secundária , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Suínos , Células Vero , Vacinas Virais/administração & dosagemRESUMO
BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.