Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

Muñoz-González, Sara; Ruggli, Nicolas; Rosell, Rosa; Pérez, Lester Josué; Frías-Leuporeau, Maria Teresa; Fraile, Lorenzo; Montoya, Maria; Cordoba, Lorena; Domingo, Mariano; Ehrensperger, Felix; Summerfield, Artur; Ganges, Llilianne

The majority of the newborn CSFV-inoculated piglets did not clear the virus CSFV replication after perinatal inoculation of the piglets with the PR and Cat01 strains was monitored by qRT-PCR for viral RNA at weekly intervals in serum, nasal and rectal swabs and in the tonsils, thymus and bone marrow at necropsy (Fig 3) . Despite the absence of clinical signs, all of the piglets had strong viral RNA signals in their serum 7 days after inoculation. At later time points, the viral RNA load further increased and remained high until the end of the experiment or until death or euthanasia in all of the piglets infected with Cat01 and in 7 of 10 piglets infected with PR. The three remaining piglets in the PR group (pigs 1, 3 and 14) cleared the virus from their circulation by day 28 post-inoculation, as determined by real time RT-PCR ( Fig 3A) . Interestingly, the Cat01 virus-infected piglets had significantly higher CSFV RNA levels in their serum than the PR virus-infected piglets (P = 0.0002), which might indicate a lower virulence of the PR strain. Virus titration at 21 and 28 days after inoculation confirmed the qRT-PCR data, with overall higher virus titres in the serum of the Cat01-infected versus the PR-infected piglets (P = 0.006, Table 1 ). Additionally, the three PR-infected piglets that had undetectable viral RNA at day 28 post-inoculation were also negative for virus isolation already at day 21 (Table 1 and Fig 3) . However, in the two sows, the virus could not be detected in the serum, either by qRT-PCR or by virus isolation at any time during the experiment, confirming the low virulence of the two strains. Nevertheless, the dam of the Cat01-infected piglets had low levels of viral RNA detectable in the nasal and rectal swabs until the end of the experiment, while the PR-infected sow remained negative from the swabs (Fig 3B and 3C) . CSFV RNA was detected in the nasal and rectal swabs of all of the inoculated piglets until the end of the experiment or the day on which they were euthanised, except for the 3 PR-infected piglets that recovered from infection and stopped shedding the virus by day 28 (Fig 3B and 3C) . The viral RNA load in the nasal and rectal swabs from the pigs infected with the Cat01 strain was higher than for the PR-infected piglets at 14 days p.i. only. At necropsy, CSFV RNA was detected in the tonsils, thymus and bone marrow of all of the piglets infected with Cat01 strain and from the 7 PR virus-infected piglets that did not clear the virus (Fig 3D) . It is worth noting that the CSFV RNA levels detected in the tonsils, thymus and bone marrow were significantly higher in the Cat01 group than in the PR group (tonsils, P = 0.0002; thymus, P = 0.009; bone marrow, P = 0.001). Interestingly, the two sows were also qRT-PCR-positive in the tonsils (data not shown). Altogether, 85% of the piglets inoculated on the day of their birth were not capable of clearing the virus within the 4 to 6 weeks of their lifespans.