Introduction
Viruses that belong to the Paramyxoviridae family have been associated with respiratory symptoms in farm animals and may cross host species barriers (Welch et al., 2017). Porcine respirovirus 1 (PRV1), also referred to as porcine parainfluenza 1, belongs to the Respirovirus genus within the Paramyxoviridae family. While the pathogenicity of PRV1 in pigs is still unclear, it has been isolated from pigs with respiratory symptoms (Lau et al., 2013). Moreover, it has been detected in co-infections with swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV), which suggests it could also play a role within the porcine respiratory disease complex (Welch et al., 2017). However, information on the global spread and epidemiology is sparse. Apart from China (Lau et al., 2013), PRV1 was only detected in the United States of America (USA) in 2016 (Palinski et al., 2016), in Chile in 2020 (Agüero et al., 2020) and very recently in Hungary also in 2020 (Dénes et al., 2020).
Here we report the first detection of PRV1 in Germany and in the Netherlands using shotgun metagenomics sequencing (SMg) and qPCR in blood serum (BS), nasal swab (NS), and oral fluid (OF) samples. In an effort to investigate the porcine virome, we screened 53 pig samples from 29 farms using SMg within the Dutch/German border region. In five farms we detected PRV1. qPCR confirmed the presence of the virus in 2 of these farms and found an additional 6 positive farms. Phylogenetic analysis found the closest match to the first detected PRV1 strain in Hong Kong (Lau et al., 2013). The Dutch/German region represents a major area of pig farming within Europe and could provide important information on the characterization and circulation of porcine viruses, such as PRV1.
Materials and Methods
Samples were first pre-selected based on a positive qPCR result for SIV (VetMAX™-Gold SIV Detection Kit, Life Technologies) and PRRSV (Virotype, Hilden, Qiagen), which are clinically relevant porcine pathogens. Overall, 34 BS from 10 farms, 4 NS from 4 farms and 15 OF samples from 15 farms (total n=53 samples) from pigs within the Dutch/German border region were selected and screened using SMg to gain an insight into the porcine virome. Next, nucleic acids were extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), including in-column DNase digestion.
Complementary DNA for SMg analysis was synthesized, as described previously (Kafetzopoulou et al., 2018). Short-read sequencing (SRS) libraries were generated using the KAPA HyperPlus Kit (Roche, Basel, Switzerland). Additionally, viral enrichment with oligonucleotide bait probes was performed on a selected sample using the SeqCap ViroCap share developer panel (Roche, Basel, Switzerland). SRS libraries were sequenced on a NextSeq 500, generating 76 base pair-reads (Illumina, San Diego, CA, USA). Furthermore, long-read sequencing (LRS) libraries were generated using the Ligation Sequencing Kit (SQK-LSK109) (Oxford Nanopore Technologies (ONT)) and sequenced on a MinION device (ONT). Lysis buffer served as a negative control.
To obtain rapid viral detection and taxonomic identification, raw-reads were uploaded onto Taxonomer (IDbyDNA, San Francisco, CA, USA). Following the detection of PRV1, sequenced reads were first trimmed, then mapped against a PRV1 database derived from available complete or near complete genomes on GenBank (n=10, 27/08/2020) using CLC Genomics Workbench v.20.0.4 (CLC) (Qiagen, Hilden, Germany). Resulting consensus sequences had to cover >300 bp of the respective reference genome to be considered valid and were subsequently confirmed and characterized by NCBI BLAST. Assembly was only performed on the BS sample as it had a high abundance of PRV1 reads, using the best PRV1 hit as guidance on CLC. To create the PRV1 phylogenetic tree, the near-complete assembled PRV1 genome from this study and PRV1 genomes from GenBank (n=10) were aligned with MAFFT (Katoh et al., 2002). Phylogenetic trees were inferred from the alignments with the Maximum Likelihood approach implemented in RAxML v8.2.10 (Stamatakis, 2014) under the General Time Reversible (GTR-G) substitution model (Stamatakis, 2014) and bootstrap of 1000 replicates. A phylogenetic analysis of the L (RdRp Polymerase) and the F (Fusion protein) genes was also performed.
For a rapid follow-up analysis, a PRV1 specific qPCR was designed to test the 53 samples, where sufficient sample material was remaining (n=50) along with an additional 17 OF samples from 17 farms in the same region. Briefly, qPCR primers were designed by aligning the available PRV1 sequences on GenBank (n=21, 01/10/2020) using Geneious Prime software v2020.0.5. Primers targeting the nucleocapsid gene were designed: 5’-GCACCACCACCTCCTCTATT-3’, 3’-GCCAAAATGGCAGGGTTRTT-5’, probe: TGCTCTCACTCCTTTTAGAACTAAATGTG. qPCR was performed using the Brilliant II qRT-PCR Master Mix 1-Step, (Agilent Technologies, Texas, USA) with a total volume of 25 μL with the following conditions: 50°C for 45 min, 95°C for 10 min, followed by 45 cycles of 95°C for 20 s, 55°C for 60 s, and 72°C for 30 s.