Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the
control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01
strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated
with the virulent strain Margarita (secondary virus). For exclusive detection of the
Margarita strain, a specific qRT-PCR assay was designed, which proved not to have crossreactivity
with the Cat01 strain. The wild boars persistently infected with CSFV were protected
from superinfection by the virulent CSFV Margarita strain, as evidenced by the
absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and
tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only
the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in
ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the
Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently
infected animals were substantially protected from superinfection after in vitro inoculation
with the Margarita virus strain, while this virus was able to infect naive PBMCs
efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B
after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to
elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune
responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally,
a sequence analysis could not detect CSFV Margarita RNA in the samples tested from
Group B. Our results suggested that the SIE phenomenon might be involved in the evolution
and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our
knowledge, this study was one of the first showing efficient suppression of superinfection in