# Surveillance of Swine Coronaviruses in Hungarian Herds with a Newly Established Pan-Coronavirus RT-PCR System

## TL;DR

This study developed a new RT-PCR system to detect a wide range of coronaviruses in swine herds, revealing the presence of specific viruses in Hungary.

## Contribution

A novel pan-coronavirus RT-PCR system was developed with improved sensitivity and specificity for detecting diverse coronaviruses.

## Key findings

- The new RT-PCR system detected coronaviruses in swine samples with a detection limit of 5–50 copies/reaction.
- Porcine respiratory CoV/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in Hungarian swine herds.
- The study emphasized the importance of RT enzyme selection and RNase inhibitors for effective detection.

## Abstract

Due to the SARS-CoV-2 pandemic, we have become familiar with coronaviruses. These viruses have a highly diverse, rapidly changing genome that aids in widespread distribution and transmission to novel hosts. Although several simple, virus-specific tests are available to detect the most important pathogenic coronaviruses, the identification of new viruses and variants requires different approaches. In our study, we developed a broad-spectrum detection system for coronaviruses. The sensitivity of the assay was measured using ten different coronaviruses that infect humans and animals and was compared with other systems. Application of the novel method demonstrated the circulation of coronaviruses in swine herds, as well as the suitability of the established tool for their detection. The assay described here is primarily intended for research purposes and could significantly advance our understanding of the diversity and host spectrum of coronaviruses and help us prepare for the emergence of new pathogens.

The rapid evolution of coronaviruses (CoVs) requires researchers to develop specific yet broad-spectrum detection methods to monitor their constant genomic changes. The goal of the present study was to establish a current pan-coronavirus RT-PCR system capable of detecting a wide variety of CoVs and useful for the investigation of virus diversity and host spectrum. For optimization, one-step and two-step nested RT-PCRs with three RT enzymes were examined, amplifying a ~600 bp long product of the RNA-dependent RNA polymerase. As templates, the in vitro transcribed RNA of ten pathogenic CoVs (SARS-CoV, SARS-CoV-2, NL-63, OC43, feline CoV, porcine epidemic diarrhea virus or PEDV, transmissible gastroenteritis virus or TGEV, canine CoV, bat CoV, and infectious bronchitis virus) were applied instead of the often-used DNA standards. A limit of detection of 5–50 copies/reaction was achieved with a random hexamer-primed two-step RT-PCR and a touchdown cycling profile, representing a lower detection limit and higher specificity compared to previously published primer sets. Swine origin pooled samples (n = 121), collected from apparently healthy herds in Hungary, were tested with the novel RT-PCR system. Sequences of porcine respiratory CoV/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in 24 oral fluid and nasal swab pools, demonstrating the circulation of these viruses in this country, as well as the suitability of the new PCR for their detection. The results highlighted the importance of adequate RT enzyme selection and the use of RNase inhibitors in sample preparation and conservation.