A poly-uridine insertion in the 3'-untranslated region of classical swine fever virus activates immunity and reduces viral virulence in piglets.

Low virulent classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Little data is available on the molecular determinants of CSFV virulence. The aim of present study is to assess a possible role for CSFV virulence of a unique uninterrupted 36-uridine (poly-U) sequence found in the 3'-untranslated region (3'UTR) of the low virulent CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3'UTR (vPdR-36U), and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of twenty 5-day-old piglets were infected with vPdR-36U and vPdR-5U, respectively. Ten contact piglets were added to each group. Disease progression, virus replication and immune responses were monitored during 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. 90% of the piglets infected with vPdR-36U seroconverted while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient IFN-α responses in the serum after one week of infection while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first two weeks. Taken together, these data show that the 3'UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3'-untranslated region (3'UTR) plays an important role in Flavivirus replication, the present study showed for the first time that a long poly-uridine sequence acquired in the 3'UTR by an endemic CSFV isolate can activate immunity, control viral replication and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants are also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.