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.