Completion of the 5′ end of the porcine teschovirus genome reveals a functional S-segment and enables reverse genetics

Porcine teschoviruses (PTVs) are swine-specific picornaviruses associated with neurological and systemic disease, ranging from severe paralytic Teschen disease to milder forms of porcine encephalomyelitis, known as Talfan disease. Despite their global distribution and veterinary relevance, complete genomic information and molecular tools for PTVs are not available. Here, we report the first complete genome sequences of PTV-A11 (strain Dresden) and a recent PTV-A field isolate (strain Gi2020_1). Both genomes, each approximately 7.2 kb in size, include a previously uncharacterized 5′-terminal region upstream of the poly(C) tract (S-segment), thereby elucidating the complete genomic architecture of PTVs for the first time. The conserved 117–118 nucleotide S-segment closes a long-standing gap in teschovirus genomics and is essential for viral replication. Using reverse genetics, we established infectious molecular clones of both strains that recapitulate the properties of their parental strains. These systems enabled functional analyses of viral gene products, including the demonstration that the leader protein is dispensable for genome replication and morphogenesis but contributes to efficient virus growth. In addition, we developed a replication-competent subgenomic replicon and engineered fluorescent reporter viruses, including a stable mCherry-expressing virus that supports robust infection analysis and allows quantitative protein expression measurements. Together, these findings define the complete PTV genome organization and provide a versatile molecular toolbox for studying teschovirus replication, pathogenesis, and control.