Results and Discussion
In total, PRV1 was detected in 5/53 samples (9.43%) from 5 different German farms using SMg directly from the sample (Table 1). PRV1 was not detected in the negative control. Four out of the 5 PRV1 sequences (Figure 1) were closely related to the Chinese PRV1 strain S033N (GenBank accession: JX857410.1), which was first isolated from deceased pigs in Hong Kong in 2013 (Lau et al., 2013). Interestingly, phylogenetic analysis from the 3 available PRV1 strains from Hungary (Dénes et al., 2020) also showed the closest similarity to strain S033N, from partial F and L sequences (Figure S1). This could indicate the S033N strain could be the most prominent strain in Central Europe. In comparison, sample NS-2 showed the closest identity to another Chinese PRV1 strain, gd2018 (GenBank accession: MK395271.1), isolated in 2018.
The untargeted nature of SMg allows the detection of all viruses within one single assay, including unexpected viruses such as PRV1. However, challenges still exist with sensitivity (Greninger, 2018). This becomes apparent even in samples with a high quantity of PRV1 sequence reads, such as in sample BS-1. As we could not recover the complete-genome in our five samples by using a direct shotgun sequencing approach, we selected the sample with the highest PRV1 reads (BS-1) to apply viral enrichment using ViroCap, in an effort to obtain the complete-genome. ViroCap has been reported to increase sensitivity of virus detection in human samples (Wylie et al., 2018). ViroCap was subsequently applied to BS-1, which resulted in a 22.8-fold increase in PRV1 sequence reads and enabled the generation of a near-complete genome sequence (15,345 bp) (GenBank accession: MT995732) and, subsequent, phylogenetic analysis.
Following the detection of PRV1 using SMg, a rapid follow-up analysis was performed using qPCR. After performing a PRV1-qPCR on 4 of the 5 samples which had a PRV1 detection using SMg, only 1 had a Ct value lower than 30 (Table 1). A Ct value of 44 was found for sample BS-1, however this was considered negative. This could be due to primer or probe – template mismatches. Divergence of the template sequence in targeted PCR can result in limited sensitivity due to primer-binding kinetics (Quick et al., 2017). Indeed, a primer mismatch was detected for the near-complete genome (sample BS-1) in the 3’-5’ primer, as well as 3 mismatches in the probe. As few sequences are available for PRV1, viral diversity is difficult to predict leading to problems in finding a sufficient conserved region to design primers and probes.
On the other hand, 3 OF samples which were negative for PRV1 using SMg, had a positive Ct value following qPCR (Table S1) highlighting a potential increased sensitivity of qPCR. Screening of the remaining 17 OF samples from 17 farms that were solely analyzed by qPCR, revealed a further 3 PRV1 positive farms (Table S1). These findings highlight the need to obtain further PRV1 complete-genome sequences in order to refine PRV1 qPCR primers for future surveillance. As demonstrated in this study, oligonucleotide capture probes can be used to help generate near-complete genomes.
SMg was unable to detect several co-detected viral pathogens which were previously detected by qPCR prior to initial sample selection. Indeed, the PRRSV (Ct 30 and 32) and SIV (Ct 36 and 37) co-detections were unable to be recovered from the OF samples. However, SMg was able to recover part of the genome for other co-detections; PRRSV (Ct 26) was recovered from the BS sample and SIV (Ct 19 and 21) was recovered from the NS samples (Table 1). Co-infections with PRRSV and/or SIV have been reported previously (Lau et al., 2013; Welch et al., 2017) but the role of these co-infections in the porcine respiratory disease complex remains to be ascertained (Welch et al., 2017; Park et al., 2019). It must be noted that samples in this study were pooled, and as such, viruses/co-infections cannot necessarily be linked to an individual animal. Nevertheless, information gathered from pooled samples can provide data on what is circulating within a farm.
Previously detected PRV1 were obtained from OF, NS and lung samples, with the upper respiratory tract being suggested to be the most suitable sampling site for detection (Lau et al., 2013; Park et al., 2019; Agüero et al., 2020). We detected the highest PRV1 read count in a BS sample, which suggests that BS could be evaluated as a possible suitable sample matrix to screen for PRV1 in the future.
In conclusion, to the best of our knowledge, we report the first detection of PRV1 in Germany and in the Netherlands, as well as the first near-complete genome in Europe. Moreover, this is the first detection of PRV1 using an untargeted and targeted metagenomic sequencing approach directly from the sample. As the PRV1 sequences from Hungary and Germany were closely related to strains previously found in China, it suggests that there may have been a PRV1 transmission between Europe and China. Furthermore, as PRV1 was detected in pigs from 11 different farms (5 using SMg and 6 using qPCR), it could confirm its circulation in Central Europe. Additional research is required to determine the extent of dissemination of PRV1 in Europe, to determine the relevance of this virus in the porcine respiratory disease complex and its ability to cross host species barriers.