**Development of a High-Throughput Serum Neutralization Test Using Recombinant Pestiviruses Possessing a Small Reporter Tag**

**Madoka Tetsuo 1, Keita Matsuno 1,2, Tomokazu Tamura 3,4, Takasuke Fukuhara 3, Taksoo Kim 1, Masatoshi Okamatsu 1, Norbert Tautz 5, Yoshiharu Matsuura <sup>3</sup> and Yoshihiro Sakoda 1,2,\***


Received: 28 January 2020; Accepted: 3 March 2020; Published: 4 March 2020

**Abstract:** A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections.

**Keywords:** border disease; bovine viral diarrhea; classical swine fever; pestivirus; serum neutralization test; reporter virus
