Mutations abrogating the RNase activity in glycoprotein E-rns of the pestivirus classical swine fever virus lead to virus attenuation

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV), Amino acid exchanges or deletions intr oduced by site-directed mutagenesis into the putative active site of the RN ase residing in the glycoprotein E-rns CSFV abolished the enzymatic activit y of this protein, as demonstrated with an RNase test suitable for detectio n of the enzymatic activity in crude cell extracts, Incorporation of the al tered sequences into an infectious CSFV clone resulted in recovery of viabl e viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. The se RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable fo r at least seven passages. In contrast to animals inoculated with an RNase- positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cel ls, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchange s of both codons 297 and 346 showed that all these RNase-negative mutants w ere attenuated. All viruses with mutations affecting codon 346 were complet ely apathogenic, whereas those containing only changes of codon 297 consist ently induced clinical symptoms for several days, followed by sudden recove ry. Analyses of reisolated viruses gave no indication for the presence of r evertants in the infected animals.