\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{First detection of porcine respirovirus 1 in Germany and in the
Netherlands}



\author[1]{Leonard Schuele}%
\author[1]{Erley Lizarazo}%
\author[1]{Hayley Cassidy}%
\author[2]{Katrin Strutzberg-Minder}%
\author[2]{Jan Boehmer}%
\author[3]{Sabine Schuetze}%
\author[3]{Sandra Loebert}%
\author[3]{Claudia Lambrecht}%
\author[3]{Juergen Harlizius}%
\author[1]{Alex Friedrich}%
\author[4]{Silke Peter}%
\author[1]{John Rossen}%
\author[1]{Natacha Couto}%
\affil[1]{University Medical Centre Groningen}%
\affil[2]{IVD Innovative Veterinnary Diagnostics}%
\affil[3]{Animal Health Services, Chamber of Agriculture of North Rhine-Westphalia}%
\affil[4]{University of Tuebingen, Institute of Medical Microbiology and Hygiene}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
PRV1 was first detected in deceased pigs from Hong Kong in 2013. It has
since been detected in the USA, Chile and most recently in Hungary.
Information on the pathogenicity and global spread is sparse, however it
has been speculated to play a role in the porcine respiratory disease
complex. 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. 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.
Together with the recent detection of PRV1 in Hungary, these findings
suggest widespread of PRV1 in Central Europe, highlighting the need for
further research on persistence, pathogenicity and transmission in
Europe.%
\end{abstract}%



\sloppy


\textbf{First detection of porcine respirovirus 1 in Germany and in the
Netherlands}

Leonard Schuele\textsuperscript{1,2}, Erley Lizarazo\textsuperscript{1},
Hayley Cassidy\textsuperscript{1}, Katrin
Strutzberg-Minder\textsuperscript{3}, Jan Boehmer\textsuperscript{3},
Sabine Schuetze\textsuperscript{4}, Sandra Loebert\textsuperscript{4},
Claudia Lambrecht\textsuperscript{4}, Juergen
Harlizius\textsuperscript{4}, Alex W. Friedrich\textsuperscript{1},
Silke Peter\textsuperscript{2}, John W. A.
Rossen\textsuperscript{1,5,+}and Natacha Couto\textsuperscript{1,6,+,*}

\textsuperscript{1} University of Groningen, University Medical Center
Groningen, Department of Medical Microbiology and Infection Prevention,
Groningen, The Netherlands

\textsuperscript{2} Institute of Medical Microbiology and Hygiene,
University of T\selectlanguage{ngerman}übingen, Tübingen, Germany

\textsuperscript{3} IVD Innovative Veterinary Diagnostics (IVD GmbH),
Seelze, Germany

\textsuperscript{4} Animal Health Services, Chamber of Agriculture of
North Rhine-Westphalia, Bad Sassendorf, Germany

\textsuperscript{5} Department of Pathology, University of Utah School
of Medicine, Salt Lake City, UT, United States of America

\textsuperscript{6} Milner Centre for Evolution, Department of Biology
and Biochemistry, University of Bath, Bath, United Kingdom

\selectlanguage{english}\textsuperscript{+}John W. A. Rossen and Natacha Couto contributed
equally.

\textbf{*Corresponding author} : Natacha Couto;\emph{nmgdc20@bath.ac.uk}
or\emph{n.monge.gomes.do.couto@umcg.nl}

\textbf{Running title:} PRV1 in Central Europe

\textbf{Summary}

PRV1 was first detected in deceased pigs from Hong Kong in 2013. It has
since been detected in the USA, Chile and most recently in Hungary.
Information on the pathogenicity and global spread is sparse, however it
has been speculated to play a role in the porcine respiratory disease
complex.

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. 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.
Together with the recent detection of PRV1 in Hungary, these findings
suggest widespread of PRV1 in Central Europe, highlighting the need for
further research on persistence, pathogenicity and transmission in
Europe.

\textbf{Keywords} : Porcine parainfluenza 1, Porcine respirovirus 1,
Europe, Germany, the Netherlands, Shotgun metagenomics sequencing

\textbf{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 (Aguero et al., 2020) and very recently in
Hungary also in 2020 (Denes 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\selectlanguage{english}-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 \textgreater{}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 \selectlanguage{greek}μ\selectlanguage{english}L with the following conditions: 50\selectlanguage{ngerman}°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.

\textbf{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.

\textbf{Data availability}

The near-complete PRV1 sequence has been deposited under the GenBank
accession: MT995732.

\textbf{Conflicts of interest}

John W. A. Rossen is employed by IDbyDNA. Silke Peter consults for
IDbyDNA. This did not influence the interpretation of reviewed data and
conclusions drawn nor on the drafting of the manuscript, and no support
was obtained from them. All other authors declare no conflict of
interest.

\textbf{Acknowledgments}

We thank Erwin C. Raangs for expert technical support and the farmers
for their participation in the study.

The Food Pro-tec-ts project is carried out within the framework of the
INTERREG V A programme ''Deutschland-Nederland'' and is co-financed by
the European Regional Development Fund (ERDF) and the Dutch Ministry of
Economic Affairs and Climate Change, the Ministry of Economic Affairs,
Innovation, Digitalisation and Energy of North Rhine-Westphalia and the
provinces of Gelderland, Limburg and North Brabant. It is accompanied by
the programme management at the Euregio Rhein-Waal. LS and HC received a
grant from the Marie Sk\selectlanguage{polish}ł\selectlanguage{english}odowska-Curie Actions (Grant Agreement number:
713660 -- PRONKJEWAIL -- H2020 -- MSCA-COFUND-2015). The funders had no
role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.

\textbf{Ethical Approval}

The animal samples used for this study were collected within the Food
Pro-tec-ts project which has been classified as an animal study and was
approved on the 22.09.2017 by the respective state office for nature,
environment and consumer protection (file reference:
84.02.05.40.17.079).

References

Ag\selectlanguage{ngerman}üero, B., Mena, J., Berrios, F., Tapia, R., Salinas, C., Dutta, J.,
van Bakel, H., Mor, S.K., Brito, B., Medina, R.A., Neira, V. (2020).
First report of Porcine Respirovirus 1 in South America.\emph{Veterinary
Microbiology} , 246,
108726.\emph{https://doi.org/10.1016/j.vetmic.2020.108726}

Dénes, L., Cságola, A., Schönhardt, K., Halas, M., Solymosi, N., Balka,
G. (2020). First report of porcine parainfluenza virus 1 (species
Porcine respirovirus 1) in Europe. \emph{Transboundary and Emerging
Diseases} , 00:1-5.\emph{https://doi.org/10.1111/tbed.13869}

Greninger, A.L. (2018). The challenge of diagnostic
metagenomics.\emph{Expert Review of Molecular Diagnostics} , 18(7),
605-615.\emph{https://doi.org/10.1080/14737159.2018.1487292}

Kafetzopoulou, L.E., Efthymiadis, K., Lewandowski, K., Crook, A.,
Carter, D., Osborne, J., Aarons, E., Hewson, R., Hiscox, J.A., Carroll,
M.W., Vipond, R., Pullan, S.T. (2018). Assessment of metagenomic
Nanopore and Illumina sequencing for recovering whole genome sequences
of chikungunya and dengue viruses directly from clinical
samples.\emph{Eurosurveillance} , 23(50), 1800228.
https://doi.org/10.2807/1560-7917.ES.2018.23.50.1800228

Katoh, K., Misawa, K., Kuma, K., Miyata, T. (2002). MAFFT: a novel
method for rapid multiple sequence alignment based on fast Fourier
transform. \emph{Nucleic Acids Research} , 30(14),
3059--3066.\emph{https://doi.org/10.1093/nar/gkf436}

Lau, S.K.P., Woo, P.C.Y., Wu, Y., Wong, A.Y.P., Wong, B.H.L., Lau,
C.C.Y., Fan, R.Y.Y., Cai J.P., Tsoi, H.W., Chan, K.H., Yuen, K.Y.
(2013). Identification and characterization of a novel paramyxovirus,
porcine parainfluenzavirus 1, from deceased pigs. \emph{Journal of
General Virology} , 94,
2184--2190.\emph{https://doi.org/10.1099/vir.0.052985-0}

Palinski, R.M., Chen, Z., Henningson, J.N., Lang, Y., Rowland, R.R.R.,
Fang, Y., Prickett, J., Gauger, P.C., Hause, B.M. (2016). Widespread
detection and characterization of porcine parainfluenza virus 1 in pigs
in the USA. \emph{Journal of General Virology} , 97,
281--286.\emph{https://doi.org/10.1099/jgv.0.000343}

Park, J.Y., Welch, M.W., Harmon, K.M., Zhang, J., Piñeyro, P.E., Li, G.,
Hause, B.M., Gauger, P.C. (2019). Detection, isolation, and \emph{in
vitro} characterization of porcine parainfluenza virus type 1 isolated
from respiratory diagnostic specimens in swine. \emph{Veterinary
Microbiology} , 228,
219--225.\emph{https://doi.org/10.1016/j.vetmic.2018.12.002}

Quick, J., Grubaugh, N.D., Pullan, S.T., Claro, I.M., Smith, A.D.,
Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T.F.,
Beutler, N.A., Burton, D.R., Lewis-Ximenez, L.L., de Jesus, J.G.,
Giovanetti, M., Hill, S.C., Black, A., Bedford, T., Carroll, M.W.,
Nunes, M., Alcantara Jr., L.C., Sabino, E.C., Baylis, S.A., Faria, N.R.,
Loose, M., Simpson, J.T., Pybus, O.G., Andersen, K.G., Loman, N.J.
(2017). Multiplex PCR method for MinION and Illumina sequencing of Zika
and other virus genomes directly from clinical samples. \emph{Nature
Protocols} , 12(6),
1261-1276.\emph{https://doi.org/10.1038/nprot.2017.066}

Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis
and post-analysis of large phylogenies. \emph{Bioinformatics} , 30,
1312-1313.\emph{https://doi.org/10.1093/bioinformatics/btu033}

Welch, M., Park, J., Gauger, P., Harmon, K., Lin, K., Pineyro, P.,
Zhang, J. (2017). Porcine Parainfluenza Virus Type 1 (PPIV-1) in US
Swine: Summary of Veterinary Diagnostic Laboratory Data. \emph{Iowa
State University Animal Industry Report} ,
14(1).\emph{https://doi.org/10.31274/ans\_air-180814-389}

Wylie, K.M., Wylie, T.N., Buller, R., Herter, B., Cannella, M.T.,
Storch, G.A. (2018). Detection of viruses in clinical samples by use of
metagenomic sequencing and targeted sequence capture. \emph{Journal of
Clinical Microbiology} , 56, e01123-18.\emph{https://doi.org/10.1128/JCM
.01123-18}

\textbf{Table 1.} Characterization of SMg-PRV1 positive samples.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Sample \& platform}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Best BLAST reference (length)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Genome} \textbf{coverage (\%)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Identity (\%)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Average sequence} \textbf{coverage}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{PRV1 qPCR}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Co-detection} \textbf{(qPCR)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Date of sampling}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Country of Origin}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
BS\textsuperscript{1}-1-Illumina\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SN0330 (15,396 bp)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
99.7\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
96.0\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
9,793\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Ct 44\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
PRRSV\textsuperscript{4} (Ct 26)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
10/2018\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Germany\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
OF\textsuperscript{2}-1-ONT\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SN0330 (15,396 bp)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
33.0\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
96.2\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
- \textsuperscript{6}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SIV\textsuperscript{5} (Ct 36) PRRSV\textsuperscript{4} (Ct 30)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
06/2017\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Netherlands\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
OF\textsuperscript{2}-2-Illumina\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SN0330 (15,396 bp)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
5.5\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
89.5\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Ct 29\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
PRRSV\textsuperscript{4} (Ct 32) SIV\textsuperscript{5} (Ct 37)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
06/2017\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Netherlands\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
NS\textsuperscript{3}-1-ONT\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SN0330 (15,396 bp)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
7.6\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
95.1\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Negative\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SIV\textsuperscript{5} (Ct 21)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
10/2018\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Germany\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
NS\textsuperscript{3}-2-Illumina\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Gd2018 (15,396 bp)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
2.4\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
97.0\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Negative\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
SIV\textsuperscript{5} (Ct 19)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
10/2018\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Germany\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{1}BS, blood serum; \textsuperscript{2}OF, oral
fluid;\textsuperscript{3}NS, nasal swab; \textsuperscript{4}Insufficient
nucleic acids left for qPCR; \textsuperscript{5}PRRSV, porcine
reproductive and respiratory syndrome virus; \textsuperscript{6}SIV,
swine influenza virus.

\selectlanguage{english}
\FloatBarrier
\end{document}

