Semi‐quantitative duplex RT‐PCR reveals the low occurrence of Porcine Pegivirus and Atypical Porcine Pestivirus in diagnostic samples from the United States

Summary Porcine Pegivirus (PPgV) and Atypical Porcine Pestivirus (APPV) are two recently identified porcine viruses. In this study, the identification of two viruses by metagenomic sequencing, and a duplex semi‐quantitative RT‐PCR was developed to detect these pathogens simultaneously. The PPgV strain Minnesota‐1/2016 had a 95.5%–96.3% nucleotide identity and clustered with the recently identified US PPgV strains, which is a distant clade from the German PPgV strains. The APPV strain Minnesota‐1/2016 shared an 87.3%–92.0% nucleotide identity with the other global APPV strains identity but only shared an 82.8%–83.0% nucleotide identity with clade II consisting of strain identified in China. Detection of both PPgV and APPV was 9.0% of the diagnostic cases. Co‐infection of PPgV and APPV was identified in 7.5% of the diagnostic cases. The occurrence and genetic characterization of PPgV and APPV further enhance our knowledge regarding these new pathogens in the United States.

Our study identified PPgV and APPV by NGS, leading to the development a semi-quantitative RT-PCR (sqRT-PCR) for these viruses to determine the occurrence of PPgV and APPV in diagnostic samples from the US. The PPgV and APPV strains from the NGS samples were phylogenetically compared to the globally available strains.

| MATERIAL SANDME THODS
During August-September of 2016, two cases were submitted to the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) to determine the causative agent of disease. Case 1 consisted of 10 serum samples from a sow farm in Minnesota experiencing an increasing incidence of mummified and stillborn foetuses.
The case was negative for routinely tested porcine pathogens. Case 2 consisted of oral fluid and was selected for NGS to discover new pathogens. The samples from these two cases were processed and submitted for library preparation and NGS on the Illumina MiSeq platform, using previously described methods (Jarvis et al., 2016).
Nucleotide and amino acid alignments were constructed using MAFFT in Geneious v9.1.8, and phylogenetic trees were built using PMYML with a GTR substitution model and bootstrapped with 500 replicates.
A duplex semi-quantitative RT-PCR (sqRT-PCR) was developed for simultaneous detection of PPgV and APPV. The PPgV primers and probe were designed based on the available PPgV strains  (Table S1) utilized the TaqMan Fast Virus 1-Step Master Mix using recommended guidelines (Thermo Fisher, Austin, TX) and 8 μl of RNA. PCR was run in the 7500 Fast Real-Time PCR System (Thermo Fisher) with the following thermal cycling conditions: reverse transcription (RT), 50°C for 5 min; RT inactivation/initial denaturation, 95°C for 20 s; followed by 40 cycles of denaturation at 95°C for 3 s and annealing at 60°C for 30 s. The analytical sensitivity was determined using custom gBlock (IDT, Skokie, IL) containing the PPgV and APPV targets while the analytical specificity was determined with 58 swine pathogens ( Figure S1). The duplex sqRT-PCR was used to test additional clinical diagnostic cases of serum or tissue from US swine herds, which were submitted to the UMVDL for routine diagnostics or surveillance for common porcine pathogens.

| RE SULTS
In case 1, 10 serum samples were negative for routinely tested porcine pathogens by PCR and viral isolation, and PPgV was the only virus detected in two of the 10 samples (samples 4 and 6), using NGS. Only 173 pegivirus reads were identified in sample 4, whereas 509 reads were identified as pegivirus in sample 6.
Using de novo assembly methods of the PPgV reads from sample 6, two PPgV contigs were obtained and was closed using  (Table 1). In phylogenetic tree, Minnesota-1 strain clustered with other US strains (Group II), which was divergent from the previously reported German PPgV strains (Group 1) (Figure 1a).
In case 2, APPV was the only pathogenic virus detected. The APPV-specific reads were de novo assembled, and a single contig representing the APPV genome of 11,397 nucleotides was generated (APPV/Pig-wt/USA/Minnesota-1/2016). The APPV strain

| D ISCUSS I ON
The However, the diversity of APPV in greater China since the strains were in different groups of the phylogenetic tree.
In this study, a duplex sqRT-PCR was developed to determine the occurrence of PPgV and APPV infections in diagnostic samples from US swine farms, following the discovery of these pathogens using NGS. A sqRT-PCR for PPgV has yet to be described (Baechlein et al., 2016;Yang et al., 2018),

CO N FLI C TO FI NTE R E S TS TATE M E NT
The authors declare no conflict of interest.

ACK N OWLED G EM ENTS
The  TA B L E 2 sqRT-PCR results for PPgV and APPV organized by specimen and state