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

Madoka Tetsuo,Norbert Tautz,Yoshiharu Matsuura,Takasuke Fukuhara,Taksoo Kim,Yoshihiro Sakoda,Tomokazu Tamura,Keita Matsuno,Masatoshi Okamatsu

doi:10.3390/pathogens9030188

Madoka Tetsuo, Norbert Tautz + Show 7 more

Open Access

https://doi.org/10.3390/pathogens9030188

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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.

Highlights

Pestiviruses are enveloped positive-strand RNA viruses that belong to the genus Pestivirus, within the family Flaviviridae
To compare the growth of recombinant vBDV FNK/High-Affinity NanoBiT (HiBiT) with the wild-type border disease virus (BDV), swine kidney line-L (SK-L) cells were inoculated with 200 50% tissue-culture infective doses (TCID50 ) of either virus
Recombinant pestiviruses were employed to establish a novel serum neutralization test (SNT) based on the luciferase system, which allows virus growth to be and rapidly monitored and enables SNT titers for pestiviruses to be determined

Summary

Introduction

Pestiviruses are enveloped positive-strand RNA viruses that belong to the genus Pestivirus, within the family Flaviviridae. Pestiviruses possess a single-stranded positive-sense RNA of approximately 12.3 kb in length, with one large open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. The ORF encodes a single polyprotein cleaved by cellular and viral proteases co- and post-translationally into four structural proteins (C, Erns , E1, and E2) and eight non-structural proteins The genus Pestivirus currently comprises 11 species, Pestivirus A to Pestivirus K, with bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV) classified into Pestivirus A (BVDV-1), Pestivirus B (BVDV-2), Pestivirus C (CSFV), and Pestivirus D (BDV), respectively [3]. The taxonomic classification of pestiviruses was based on the host species from which they were isolated (e.g., CSFV from pigs and BVDV from cattle), but it is well known that many pestiviruses are capable of interspecies transmission (e.g., BVDV infections in pigs and BDV infections in cattle) [4,5]

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