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.IMPORTANCETeschen disease was once a devastating neurological disease of swine caused by highly virulent porcine teschovirus strains (PTVs) but has become rare following their eradication. Current control strategies base on hygiene measures and herd-specific vaccination providing limited protection and leaving swine populations vulnerable to the (re-)emergence of neuroinvasive PTVs. Less virulent strains remain endemic worldwide and continue to impair animal health, welfare, and production efficiency. Progress toward broadly effective vaccines and antivirals has been constrained by incomplete knowledge of the PTV genome. Here, we identify and functionally define the previously unrecognized S-segment that completes the 5' UTR of PTVs. We further established infectious cDNA clones and developed subgenomic replicons, leaderless viruses, and fluorescent reporter viruses. Our tools enable direct genetic manipulation of PTVs. They provide a platform for mechanistic studies of viral replication, attenuation, and antigen design supporting rational development of vaccines and antiviral strategies against emerging PTVs.