Escape of classical swine fever C-strain vaccine virus from detection by C-strain specific real-time RT-PCR caused by a point mutation in the primer-binding site.

Classical swine fever (CSF) is one of the most important diseases of pigs. Vaccination in the European Union is limited to emergency situations. Currently, vaccination for the purpose of disease control is carried out in wild boar populations. Wild boar are in most cases vaccinated using an oral bait vaccine based on the live modified vaccine virus C-strain "Riems". A real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for differentiation of C-strain "Riems" vaccine virus from CSF virus (CSFV) field isolates was published previously. In this real-time RT-PCR system differentiation is based on two nucleotide difference one at the 3' end of each of the primer-binding sites in the E(RNS) encoding genome region. During extensive diagnostic use of this protocol in an outbreak of CSF in wild boar in Germany, some C-strain positive field samples were found to give negative results in the C-strain "Riems" specific real-time RT-PCR, but positive results in a pan-CSFV real-time RT-PCR system. Moreover, sequencing of C-strain "Riems" vaccine batches for intramuscular use revealed differences in the E(RNS) encoding region. This led to the assumption that mutations in the corresponding primer-binding site of the C-strain specific system had appeared in the field, and possibly also during manufacturing of different vaccine batches. To test this hypothesis and restore sensitivity, a new primer set for detection of the possible C-strain virus quasi species was designed and tested.