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\begin{document}

\title{A single-tube triplex real-time quantitative PCR assay for differential
detection of highly virulent Chinese strains of pseudorabies virus}



\author[1]{Orlando Perez}%
\author[1]{Mathieu Pinette}%
\author[2]{Jianfa Bai}%
\author[3]{Lalitha Peddireddi}%
\author[4]{John  Schiltz }%
\author[4]{Karthik Shanmuganatham}%
\author[4]{Rachel Tell}%
\author[5]{Alfonso Clavijo}%
\author[1]{Aruna Ambagala}%
\affil[1]{Canadian Food Inspection Agency}%
\affil[2]{Kansas State University}%
\affil[3]{Kansas State Veterinary Diagnostic Laboratory, KansasState University}%
\affil[4]{USDA National Veterinary Services Laboratories}%
\affil[5]{USDA}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Pseudorabies virus (PRV) causes Aujeszky's disease or pseudorabies (PR),
which is characterized by fatal encephalitis in newborn piglets,
respiratory infection in growing and fattening pigs, and reproductive
failures in pregnant sows. It establishes a lifelong latent infection in
the peripheral nervous system followed by subsequent intermittent
shedding of infectious virus. Since 2011, highly virulent PRV strains
that are genetically different from the classic PRV strains surfaced in
pig herds in China. Availability of a highly sensitive and specific
polymerase chain reaction (PCR)-based diagnostic assay for rapid
differential detection of PRV variants is critical to prevent huge
economic losses to the U.S. and Canadian pork industries if these
strains enter North America and cause an outbreak. Here we describe the
development and evaluation of a single-tube triplex real-time-PCR assay
for differential detection of variant strains of PRV. The assay targets
the intergenic region between the US2 and US6 genes in the PRV genome,
is highly sensitive and specific, and it did not detect other non-target
viruses, including related herpesviruses. The clinical specificity and
sensitivity of the assay was evaluated using whole blood, serum, tissue
and swab samples collected from known negative and experimentally
inoculated pigs with either classical (Bristol) or variant (JS-2012 and
HeN1) PRV strains. The targeted genomic region of this assay is also
deleted in commonly used PRV gE-deleted marker vaccines, and therefore,
the triplex assay did not detect viral DNA extracted from two commercial
vaccine strains Bartha K-61 and Bucharest. This single-tube triplex
assay can be used for routine diagnostics and epidemiological studies
for detection and differentiation of classical strains from variant
strains of PRV, and as a differentiation of infected and vaccinated
animals (DIVA) assay when PRV gE- deletion mutant marker vaccines are
used.%
\end{abstract}%



\sloppy


Orlando Perez\textsuperscript{1}, Mathieu Pinette\textsuperscript{1},
Jianfa Bai\textsuperscript{4}, Lalitha Peddireddi\textsuperscript{4},
John Schiltz\textsuperscript{5}, Karthik,
Shanmuganatham\textsuperscript{5}, Rachel M. Tell\textsuperscript{5},
Alfonso Clavijo\textsuperscript{1,+}, Aruna Ambagala\textsuperscript{1,
\#}

\textsuperscript{1}National Centre for Foreign Animal Disease, Canadian
Food Inspection Agency, Winnipeg, MB, Canada.\textsuperscript{2}Shanghai
Veterinary Research Institute, Chinese Academy of Agricultural Sciences,
Shanghai, China.\textsuperscript{3}College of Veterinary Medicine,
Northwest A \& F University, Yangling, China. \textsuperscript{4}College
of Veterinary Medicine, Kansas State University, Kansas, KS,
USA.\textsuperscript{5}National Veterinary Services Laboratories, VS,
APHIS, USDA, Ames, IA, USA.

\textsuperscript{\#} Corresponding author

+ Current address: USDA, Agricultural Research Service, Manhattan, KS,
USA

\textbf{Abstract}

Pseudorabies virus (PRV) causes Aujeszky's disease or pseudorabies (PR),
which is characterized by fatal encephalitis in newborn piglets,
respiratory infection in growing and fattening pigs, and reproductive
failures in pregnant sows. It establishes a lifelong latent infection in
the peripheral nervous system followed by subsequent intermittent
shedding of infectious virus. Since 2011, highly virulent PRV strains
that are genetically different from the classic PRV strains surfaced in
pig herds in China. Availability of a highly sensitive and specific
polymerase chain reaction (PCR)-based diagnostic assay for rapid
differential detection of PRV variants is critical to prevent huge
economic losses to the U.S. and Canadian pork industries if these
strains enter North America and cause an outbreak. Here we describe the
development and evaluation of a single-tube triplex real-time-PCR assay
for differential detection of variant strains of PRV. The assay targets
the intergenic region between the US2 and US6 genes in the PRV genome,
is highly sensitive and specific, and it did not detect other non-target
viruses, including related herpesviruses. The clinical specificity and
sensitivity of the assay was evaluated using whole blood, serum, tissue
and swab samples collected from known negative and experimentally
inoculated pigs with either classical (Bristol) or variant (JS-2012 and
HeN1) PRV strains. The targeted genomic region of this assay is also
deleted in commonly used PRV gE-deleted marker vaccines, and therefore,
the triplex assay did not detect viral DNA extracted from two commercial
vaccine strains Bartha K-61 and Bucharest. This single-tube triplex
assay can be used for routine diagnostics and epidemiological studies
for detection and differentiation of classical strains from variant
strains of PRV, and as a differentiation of infected and vaccinated
animals (DIVA) assay when PRV gE- deletion mutant marker vaccines are
used.

\textbf{Keywords:}

Pseudorabies Virus, Aujeszky's, Variant, Real-time quantitative PCR,
Triplex PCR, detection, differentiation, DIVA

\textbf{Introduction:}

Pseudorabies virus (PRV), or suid herpesvirus-1, is a double stranded
DNA virus of the genus \emph{Varicellovirus} ,
subfamily\emph{Alphaherpesvirinae} , and family \emph{Herpesviridae}
(Lee and Wilson, 1979; Mettenleiter, 2000; Pomeranz et al., 2005). It
causes pseudorabies (PR) or Aujeszky's disease in livestock and wild
animals including ruminants, carnivores and rodents; however, pigs are
the primary host and reservoir for this virus. PRV causes fatal
encephalitis in newborn piglets, respiratory signs in growing and
fattening pigs, and reproductive failures in pregnant sows. Like other
herpesviruses, PRV establishes a lifelong latent infection in the
peripheral nervous system, which increases the difficulty in eradicating
this virus (Gu et al., 2015).

Pseudorabies has spread throughout the world, but Canada, Greenland, and
Australia are considered free of this disease. PRV emerged as a
significant pathogen in the USA in the 1960s as a result of emergence of
virulent strains or the increase in confinement swine housing. In 2004,
PRV was eradicated from the U.S. commercial swine herds, but the virus
remains in some localized feral swine populations (United States
Department of Agriculture, 2008). The presence of PRV in U.S. feral
swine poses an on-going threat to the North American swine industry
(Hahn et al., 2010; Gaskamp et al., 2016). Pseudorabies is a reportable
disease in the U.S. and Canada.

China is considered the largest pork producer in the world. The earliest
documented PRV outbreak in China was in 1947. Since the 1990s, more than
80\% of pigs in China have been vaccinated with Bartha-K61 vaccine; and
PR has been well controlled. In late 2011, a newly emerged PRV variant
with clinical manifestations of high fever, depression, anorexia, cough,
shivering, diarrhea, and systemic neurological symptoms with high
mortality surfaced in Bartha-K61 vaccinated pig herds in Northern China
(An et al., 2013; Hu et al., 2015a, Hu et al., 2015b; Tong et al., 2015;
Yang et al., 2016; Yu et al., 2014). Since then, this virus has spread
across China causing severe economic losses. These new PRV variants when
inoculated into na\selectlanguage{ngerman}ïve pigs show earlier onset of clinical signs, severe
pathological lesions in the brains (subarachnoid hemorrhages, cerebral
edema), lungs (pulmonary congestion and edema) and higher mortality
compared to other PRV strains. Partial and whole genome analyses of
these viruses show many amino acid mutations and insertions in
functionally important structural (gE, gC, UL36, etc.) and
non-structural (US1) virial proteins (Lee and Wilson, 1979; Luo et al.,
2014; Xiang et al., 2016; Yoon et al., 2005). Based on phylogeny, these
newly identified highly virulent PRV variants from China have been
assigned to Genotype II, whereas PRV strains from other countries, or
classical strains, are assigned to Genotype I (Gu et al., 2015).

The gold standard of PR laboratory diagnosis is virus isolation,
however, it is labor intensive and time consuming. Alternatively,
polymerase chain reaction (PCR) assays (conventional and real-time) can
rapidly detect PRV DNA in nasal and oropharyngeal swabs and organ
samples from infected animals. The PCR-based assays are highly
sensitive, and they can even detect viral DNA in latently infected
animals from which virus can be difficult to isolate.

Conventional PCR assays require electrophoresis of the PCR products at
the end of the amplification, and, therefore, are time consuming,
labor-intensive, and cannot be automated. In contrast, real-time
quantitative PCR (qPCR) assays allow detection of PCR amplification
during early phases of amplification, are quantifiable, and can be
automated. A number of qPCR assays for detection of classical PRV
strains (pan-PRV assays) have been developed. The majority of these are
TaqMan probe-based assays (Ma et al., 2008; van Rijn et al., 2004;
Wernike et al., 2014; Wu et al., 2014; Zhang et al., 2015; Zhao et al.,
2008), while a few were intercalating dye-based (SYBR® Green/EvaGreen®)
assays (Perez et al., 2012; Rao et al., 2014). The probe-based assays
are more specific and therefore preferred over dye-based assays. A
TaqMan probe-assay has been validated by the OIE for use in surveillance
and routine diagnosis by local veterinary laboratories. This assay
(ADIAVET (®) PRV REALTIME from Adiagène, Saint-Brieuc, France) targets
the PRV gD glycoprotein-encoding gene and is commercially available as a
duplex assay containing an exogenous positive control (Pol et al.,
2013). The United States Department of Agriculture's (USDA) Agricultural
Research Service has also developed a set of real-time PCR assays, which
can be multiplexed and can differentiate wildtype classical PRV strains
from commercial gE-deleted modified live virus marker vaccines (Zanella
et al., 2012). None of these assays, however, can differentiate pigs
infected with highly pathogenic PRV strains (Genotype II) from those
infected with classical (Genotype I) viruses.

Highly pathogenic variant PRV strains, if they enter North America,
could cause huge economic losses to the U.S. and Canadian pork
industries. Availability of a rapid, highly-sensitive, PCR-based
diagnostic assay for simultaneous detection and differentiation of PRV
variants could be critical to prevent such losses. The currently
available vaccines in North America for PRV are based on classical
(Genotype I) PRV strains, and they may not provide complete protection
against highly virulent Chinese strains (Genotype II) (Yu et al., 2014,
An et al., 2013, Wang et al., 2014, Wang et al., 2018). As a result it
is critical to rapidly detect and differentiate the highly pathogenic
Chinese strains if they spread to North America.

Here we describe development and evaluation of a novel single-tube
triplex qPCR assay with a built-in internal control for rapid and
simultaneous detection and differentiation of highly pathogenic Genotype
II PRV strains from Genotype I strains.

\textbf{Materials \& Methods:}

\textbf{Viruses:}

Highly pathogenic PRV JS-2012 variant strain (Tong et al., 2015) was
obtained from Shanghai Veterinary Research Institute, Shanghai, China.
The classical PRV strain Bristol (classical strain), Shope, Becker, and
Bartha strains were available at the National Centre for Foreign Animal
Diseases (NCFAD)-Winnipeg. All these viruses were propagated and
titrated in PK-15 cells. The PRV modified live vaccine AUSKIPRA - GN
(PRV Bartha-K61 strain) was purchased from Laboratorios HIPRA S.A. in
Spain and PR-VAC PLUS (PRV Bucharest strain) was kindly provided by
Zoetis Inc. in Lincoln, NE, USA. PRV genomic DNA was extracted from
tissue culture amplified virus, PRV positive clinical samples, or from
vaccines reconstituted in PBS. Cross reactivity (analytical specificity
or exclusivity) of the PCR with other swine viruses was tested with a
genomic panel comprised of swine viruses extracted from cell culture
amplified African and classical swine fever viruses and foot and mouth
disease virus (FMDV); clinical samples positive for porcine respiratory
and reproductive syndrome virus (PRRSV), porcine epidemic diarrhea virus
(PEDV), and porcine circovirus 2 (PCV2); and herpesviruses originating
in bovine, elk , caprine, \emph{Rangifer} , and an unknown cervid . PRV
pUC57 plasmid (545 ng/\selectlanguage{greek}µ\selectlanguage{english}L) was purchased from Genscript.

\textbf{Primers and Probes:}

Primers and probes to detect and differentiate classical and variant PRV
strains were designed using Geneious v10.1.1 (Biomatters Inc. Newark,
NJ, USA) bioinformatics software sequence analysis tool. To identify the
target regions for the primers and probes, an alignment of 34 PRV
sequences was compiled, consisting of 12 full-length and six partial
classical strains, as well as 13 full-length and three partial strains
of variant PRV. The sequence data for these viruses were downloaded from
the NIAID Virus Pathogen Resource (ViPR - http://www.viprbrc.org) and
GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The six partial
sequences were included as they were used in the design of the triplex
assay recently reported by Meng et al., 2016. Once the sequences were
aligned, genomic regions showing multiple mutations or insertions
between PRV classical and variant strains were identified. Of those
targeted regions, the untranslated region between the US9 gene and the
US2 gene was selected (from 127,349bp to 127,485bp nucleotides in
JS-2012 PRV strain) as the preferred region for primer/probe design.
Within this region, a 21-nucleotide insertion was identified in all
variant strain sequences available, and it was used as the target for
the PRV US9/US2 UTR variant probe. Downstream of the insertion, we
identified a region that shows 13 nucleotide polymorphisms between
classical and variant PRV strains. The PRV US9/US2 UTR classical probe
was designed to capture 10 out of the 13 polymorphisms observed in this
region. It was also observed that the targeted region is deleted in
Bartha-K61 and Bucharest strains that are commonly used in commercial
PRV glycoprotein E (gE)-deleted marker vaccines (Figure 1).

Two primers, PRV US9/US2 UTR RRT Forward (5'-ACACAGCAGCCTTCCT-3') and
PRV US9/US2 UTR RRT Reverse (5'-GCGTGACCACGGTGA-3') were designed to
amplify a 137bp long PCR product from variant PRV strains and a 116bp
PCR product from classical strains. In order to differentiate classical
and variant PRV strains, two strain-specific probes, PRV US9/US2 UTR
classical and PRV US9/US2 UTR variant, were designed (Table 1A). As the
internal control, primers and a probe specific for \selectlanguage{greek}β-\selectlanguage{english}Actin (\selectlanguage{greek}β-\selectlanguage{english}Actin
1036R 831FP, \selectlanguage{greek}β-\selectlanguage{english}Actin 1036RP and \selectlanguage{greek}β-\selectlanguage{english}Actin-probe-880) were used (Moniwa et
al., 2007). For detection of all PRV strains, primers and probes from a
widely used, highly sensitive and specific PRV gB real-time PCR assay
were employed (Ma et al., 2008). To facilitate the triplex assay
development, determine specificity, and to generate positive controls
for the assay, synthetic gene fragments (gBlocks) of the target regions
of PRV classical and variant genomes and the \selectlanguage{greek}β-\selectlanguage{english}actin gene were
synthesized and procured from Integrated DNA Technologies, Inc.
(Coralville, Iowa) (Table 1B).

\textbf{Animal experiments:}

To generate clinical samples for validation of the triplex assay, a
total of 19 Landrace x large white crossbred pigs (11 piglets at three
weeks of age and eight weaner pigs at seven weeks of age) were purchased
from a commercial swine farm in Southern Manitoba and transported to the
National Centre for Foreign Animal Disease (NCFAD) animal facility.
After seven days of acclimatization, pigs were randomly assigned to
three groups: Group 1 (eight 7-weeks old pigs for PRV-JS-2012, Pig\#
5-12), Group 2 (eight 3 weeks-old piglets for PRV-JS-2012, pig \# 13-20)
and Group 3 (three 3 weeks old piglets for PRV-Bristol, Pig \# 24-26).
Each group was housed in an individual pen. Each pig in Group 1 and 2
received 2 ml of PRV Strain JS-2012 (10\textsuperscript{5} TCID50/ml, 1
ml in each nostril) and each pig in Group 3 received 2 ml of PRV Bristol
strain (10\textsuperscript{5} TCID50/ml, 1 ml in each nostril). After
the challenge, all animals were monitored twice daily for clinical signs
(fever, neurological, respiratory etc.); and oral fluid, nasal and
oropharyngeal swabs were collected every other day starting at 2 days
post infection (dpi) until 14 dpi. Pigs that reached humane endpoint
were euthanized and tissue samples (tonsils, spleen, lung, brain,
trigeminal ganglia, kidney, and lymph nodes) were collected. The
remaining animals were euthanized at 14 days post infection and similar
samples as above were collected. All the samples were stored at -80\selectlanguage{ngerman}ºC
until tested. The animal experiment was approved by the Animal Care
Committee at the Canadian Science Centre for Human and Animal Health.
The guidelines from the Canadian Council for Animal Care were observed
during all procedures with animals.

\textbf{Nucleic acid extraction:}

Total nucleic acid was extracted from 55 \selectlanguage{greek}µ\selectlanguage{english}l of each sample (cell culture
supernatant, nasal and oral swabs, serum, whole blood or 10\% tissue
suspensions) using 5X MagMAX\textsuperscript{TM} Pathogen RNA/DNA Kit on
a MagMAX Express-96 Deep well magnetic particle processor (Applied
Biosystems) following manufacturer's protocol. The nucleic acid was
eluted in 50 \selectlanguage{greek}µ\selectlanguage{english}l of elution buffer. The MagMAX-96 magnetic particle
processor was used to extract samples per appropriate script for the
sample volume tested.

\textbf{PRV triplex qPCR assay development and optimization:}

Three separate qPCR assays (classical PRV RT-PCR, variant PRV qPCR and
\selectlanguage{greek}β-\selectlanguage{english}Actin qPCR) were first developed and later combined into a triplex
assay. The triplex assay was optimized on the Applied Biosystems 7500
Real-Time PCR System using detection channels for FAM (absorbance at
495nm, emission at 520nm), HEX (absorbance at 535nm, emission at 556nm),
and Q670 (absorbance at 647-670 nm). The optimized PCR reaction (20 \selectlanguage{greek}µ\selectlanguage{english}l)
contains 5.5 \selectlanguage{greek}µ\selectlanguage{english}L sterile PCR grade water, 5 \selectlanguage{greek}µ\selectlanguage{english}L 4X TaqMan FAST Virus
1-Step Master Mix, 1 \selectlanguage{greek}µ\selectlanguage{english}L of PRV forward and 1 \selectlanguage{greek}µ\selectlanguage{english}L PRV reverse primers (0.5
\selectlanguage{greek}µ\selectlanguage{english}M final), 0.5 \selectlanguage{greek}µ\selectlanguage{english}L PRV variant probe (0.25 \selectlanguage{greek}µ\selectlanguage{english}M final), 0.5 \selectlanguage{greek}µ\selectlanguage{english}L PRV
classical probe (0.25 \selectlanguage{greek}µ\selectlanguage{english}M final), 2 \selectlanguage{greek}µ\selectlanguage{english}L of \selectlanguage{greek}β-\selectlanguage{english}Actin forward and 2 \selectlanguage{greek}µ\selectlanguage{english}L of
\selectlanguage{greek}β-\selectlanguage{english}Actin reverse primers (1 \selectlanguage{greek}µ\selectlanguage{english}M final), 0.5 \selectlanguage{greek}µ\selectlanguage{english}L of \selectlanguage{greek}β-\selectlanguage{english}Actin probe (0.2 \selectlanguage{greek}µ\selectlanguage{english}M
final) and 2 \selectlanguage{greek}µ\selectlanguage{english}L extracted nucleic acid from the sample. The optimized
triplex assay conditions are: 50\selectlanguage{ngerman}\textsuperscript{º}C for 5 minutes, 95ºC
for 20 seconds, and 45 cycles of 95ºC for 3 seconds and 60ºC for 30
seconds. All primers and probe working stocks were maintained at 10 \selectlanguage{greek}µ\selectlanguage{english}M
concentration. The primers and probe for the gB qPCR assay was adapted
from Ma et al, (2008) and the forward and reverse primers were used at
0.4 \selectlanguage{greek}µ\selectlanguage{english}l per reaction (0.4 \selectlanguage{greek}µ\selectlanguage{english}M final) with the probe at 0.4 \selectlanguage{greek}µ\selectlanguage{english}l per reaction
(0.2 \selectlanguage{greek}µ\selectlanguage{english}M final) with the stock solutions being 20 \selectlanguage{greek}µ\selectlanguage{english}M for primers and 10
\selectlanguage{greek}µ\selectlanguage{english}M for the probe. The same PCR cycling conditions were used for the
triplex and the gB assays.

\textbf{Analytical specificity, sensitivity and variability testing:}

Analytical specificity of the assay was determined using DNA extracted
from cell culture amplified PRV strains Bartha, Becker, Shope, Bristol
C61, and JS-2012, common swine viral pathogens encountered in modern
swine herds, and bovine herpesvirus-1 (BHV-1), a related herpesvirus.
For evaluating the intra-assay variability, 20 replicates of a single
sample were tested in a single run. The inter-assay variability was
calculated by running the same assay three times over 3 days. The
analytical sensitivity of the triplex assay was determined using known
concentration of PRV plasmid with net copy number of
7.34x10\textsuperscript{11}. The limit of detection is interpreted as
the lowest copy number that can be detected by the assay.

\textbf{Virus isolation}

Virus isolation was performed on PK-15 cells grown in 96-well tissue
culture plates. Briefly, PK-15 cells were seeded at 500,000 cells per
well in 96 well tissue culture plates using \selectlanguage{greek}α-\selectlanguage{english}MEM supplemented with 2 mM
L-glutamine, 5mg/mL gentamicin and 2\% \selectlanguage{greek}γ-\selectlanguage{english}irradiated Fetal Bovine Serum
(FBS). Ten percent (W/V) homogenates were prepared from each tissue and
25 \selectlanguage{greek}µ\selectlanguage{english}l of each suspension was added to each well of the PK-15 cells. A
positive control plate was inoculated with a PRV variant strain
(JS-2012). The plates were incubated for 48 hours in a 5\%
CO\textsubscript{2} humid air incubator at 37\selectlanguage{ngerman}\textsuperscript{°}C and
were checked daily for cytopathic effect (CPE) and possible
contamination using a light microscope. Two days post infection, the
cells were fixed using 200 \selectlanguage{greek}µ\selectlanguage{english}l of fixation fluid (Acetone, PBS, and
Bovine serum albumin fraction V) per well, and stained using
porcine-origin PRV antiserum. Briefly, the plates were rehydrated for 30
minutes (using 150 \selectlanguage{greek}µ\selectlanguage{english}l of PBS at room temperature), incubated with PRV
antibody positive polyclonal serum (diluted 1:100 in PBS-0.1\%Tween 20)
for 30 minutes at 37\selectlanguage{ngerman}\textsuperscript{°}C, followed by a 30-minute
incubation with rabbit anti-swine IgG HRP conjugate at
37\textsuperscript{°}C and finally for 15 minutes at
37\textsuperscript{°}C with t3-Amino-9-ethylcarbazole (AEC) substrate
solution. After the substrate incubation, the plates were washed, and
positive staining recorded under a light microscope. PRV JS-2012 and
Bristol strains with known titers were used as positive controls.

\textbf{Inter-laboratory comparison and additional characterization:}

The optimized the triplex assay along with the gB assay were transferred
to Kansas State University Veterinary Diagnostic Laboratory (KSU-VDL)
for inter-laboratory comparison using a negative cohort study comprised
of negative clinical samples collected from U.S. swine herds. A few
known-negative clinical samples were spiked with synthetic PRV-specific
DNA fragments (gBlocks) and tested on the triplex assay to mimic
positive clinical situations.

For the negative cohort study, KSU-VDL used 400 known PRV-negative
clinical samples collected from healthy pigs in the U.S. As positive
controls, 22 clinical samples (serum, oral fluids, and fecal swabs) were
spiked with PRV classical or PRV variant gBlocks. The assay was also
evaluated at the U.S. Department of Agriculture's National Veterinary
Services Laboratories (NVSL) in Ames, Iowa, for its ability to detect a
second PRV variant strain, HeN1 (An et al., 2013) using archived vaccine
challenge study samples (58 nasal swabs and 58 oral swabs), which were
collected from seven pigs vaccinated with a USDA licensed, commercially
available PRV DIVA vaccine and five unvaccinated pigs, all of which had
been challenged with the PRV HeN1 (NVSL, unpublished data).

\textbf{Results:}

\textbf{Development and optimization of a single-tube, triplex real-time
qPCR assay.}

A triplex assay that can differentiate highly virulent PRV variant
strains from classical strains was successfully designed targeting the
US2- US9 intergenic region. This region is deleted in Bartha-K61 and
Bucharest PRV strains which are commonly used in commercial PRV marker
vaccines; therefore, the assay can be used to differentiate animals
vaccinated with these vaccines from those infected with wild-type PRV
strains (DIVA assay). For the assay development, genomic DNA was
extracted from PK-15 cells infected with PRV classical strain Bristol,
PRV Variant strain JS-2012, and PRV gE- deleted DIVA vaccine strain
Bartha-K61. Prior to use for assay characterization, the sequence
identity of these three virus strains was confirmed by PCR amplification
of the region flanking the gE deleted region of the Bartha-K61 strain
using forward primer 5'-GTACCGGCGTCGATGATGAT-3'' and reverse primer
5'-GCCCAGGATCCACAGGTG-3', followed by Sanger sequencing.

The triplex assay conditions were optimized using the synthetic gene
fragments (gBlocks) resembling the target regions of PRV classical and
variant virus genomes and the \selectlanguage{greek}β-\selectlanguage{english}actin gene (data not shown) according to
the cycling conditions recommended for the TaqMan FAST Virus 1-Step
Master Mix kit. The optimized triplex assay was able to accurately
detect and differentiate the PRV classical strains Bristol, Shope and
Becker and the variant strain JS-2012. The triplex assay did not detect
any of the closely related herpesviruses or other high consequence swine
viral pathogens tested, including ASF, CSF, and FMD viruses (Table 2).
As expected, the triplex assay was unable to detect the gE-deleted PRV
Bartha-K61 Vaccine strain and the PRV Bucharest vaccine strain. The gB
assay, in contrast, detected all tested PRV strains, confirming the
presence of PRV viral DNA in these samples.

Once the triplex assay was optimized, the standard curves of the assay
were generated and PCR efficiency was determined by plotting Ct value
against log copy number from ten-fold serial dilutions of PRV plasmid
with net copy number of 7.34x10\textsuperscript{11} for both classical
and variant strains of PRV (Figure 2). The assay values with Ct values
\textgreater{}40 were not plotted on the graph. The regression line
equation for PRV Variant (FAM channel) was y = -3.3563x + 39.416 with a
correlation coefficient (R\textsuperscript{2}) of 0.9941. The regression
line equation for PRV Classical (HEX channel) was y = -3.5375x + 41.126
with a correlation coefficient (R\textsuperscript{2}) of 0.9907. The PCR
efficiencies were determined to be 98.59\% and 91.73\% for variant and
classical strain targets, respectively.

To determine the limit of detection of the assay, PRV plasmid was
diluted ten-fold to extinction. The triplex assay was able to detect as
low as 0.86 copies for PRV Variant (FAM) and 1.86 copies for PRV
Classical (HEX) targets.

The repeatability of the triplex assay was evaluated by intra-assay and
inter-assay variability testing. The coefficient of variation (CV)
within each replicate was determined as a percentage of the ratio of
standard deviation and mean of Ct values from FAM (specific for variant
strain) and HEX (specific for classical strain) channels. Inter-assay
variability testing was done with the triplex run three times over three
days with one replicate for each dilution of PRV Bristol and JS-2012
viruses. Intra-assay variability was calculated with 20 replicates in a
single run using the 10\textsuperscript{-2} dilution of PRV JS-2012 and
Bristol viruses. All variability testing was performed on the Applied
Biosystems 7500 FAST real-time PCR machine. The triplex assay
demonstrated good repeatability for the diluted samples of PRV JS-2012
and PRV Bristol with the inter-assay ranging from 0.52 [?] CV [?] 3.64 and
the intra-assay CV ranging from 1.74 [?] CV [?] 2.02 for both strains. The
diluted samples of both PRV strains were run on three different
real-time PCR instruments including the Light Cycler 480, Applied
Biosystems 7500, and Bio-Rad CFX 96. The results were consistent across
the real-time PCR platforms tested (data not shown).

\textbf{Diagnostic sensitivity and specificity of the PRV triplex
real-time qPCR}

To evaluate the diagnostic sensitivity and specificity of the PRV
triplex assay, 114 nasal swab samples were collected from pigs
experimentally inoculated with PRV JS-2012 or PRV Bristol at the
NCFAD-Winnipeg laboratory animal facility. Severe disease was expected
in pigs inoculated with the highly virulent PRV strain JS-2012 (Luoe et
al., 2014); therefore, 16 pigs representing two different age groups,
3-weeks and 7-weeks, were used for PRV JS-2012 inoculations. Out of the
eight JS- 2012 inoculated 3-week old piglets, six developed fever,
depression and respiratory signs (sneezing and nasal discharge) by 3
dpi. Two of the inoculated 3-weeks old piglets were euthanized on 5 dpi;
four were euthanized on 7 dpi due to severe neurological signs (tremors
and seizers). The remaining two piglets developed mild neurological
signs by 14 dpi and were euthanized on 16 dpi. The JS-2012 inoculated
7-week old pigs survived the infection until the end of the experiment
and were euthanized on 14 dpi. The majority of those pigs (6 of 8)
developed fever by 4 dpi, as well as mild respiratory (sneezing, nasal
discharge) and neurological signs (lameness, prostration); but by 11
dpi, they all had recovered. The three 3-week old piglets inoculated
with PRV Bristol developed clinical signs (fever, lack of appetite) by 3
dpi; and they were euthanized on 7 dpi as they had developed severe
neurological signs.

Both the PRV gB assay and the triplex assay detected PRV nucleic acid in
nasal swabs from 3-week old piglets as early as 4 dpi and starting at 6
dpi in 7-week old pigs (Table 3). Low levels of PRV nucleic acid were
detected earlier in some animals by both assays (on 4 dpi in some 7-week
old pigs and on 2 dpi in some 3-week old piglets). Of the 102 samples
tested, 62 samples were positive, 36 negative, and 4 were suspect (Ct
\textgreater{}35.99 were determined as suspect) on the triplex assay for
PRV nucleic acids. The PRV gB assay identified 57 samples as a positive,
35 samples as negative, and 10 samples as suspect. While the gB PCR as
published did not define a suspect range, for this project, we
designated Ct greater than 35.99 as suspect (Table 4).

Virus isolation was performed on a subset of samples representing
different Ct values and sample types (Table 5). PRV-like cytopathic
effect (rounding up of cells and occasional formation of syncytia) and
positive staining of cells was observed for the positive control wells,
and no staining was observed in negative wells, as expected. The Ct
values of the triplex assay and the gB assay were comparable for all the
tissue samples from PRV JS-2012 and PRV Bristol infected animals. No
cross-reactivity was observed on the triplex assay. Virus isolation was
successful from all samples with Ct values less than 30 on both the
triplex and gB assays.

\textbf{Inter-laboratory comparison and additional characterization:}

Inter-laboratory comparison of the triplex assay was performed at the
KSU Veterinary Diagnostic Laboratory and the NVSL in Ames, Iowa. At the
KSU VDL, 440 negative samples including oral fluids, serum, feces, lung,
tissue and nasal swabs collected from the U.S. commercial swine herd
were tested on the triplex real-time PCR and the gB real-time PCR (data
not shown). All clinical samples were negative for both classical and
variant PRV nucleic acid, as expected. Fluorescent signal for Q670
(endogenous internal control, \selectlanguage{greek}β-\selectlanguage{english}actin) was detected on all samples, with
Ct values ranging from 18.73 to 35.85, confirming efficient DNA
extraction and absence of PCR inhibitors in the samples (data not
shown).

As positive controls, 11 negative serum samples and one negative oral
fluid sample were spiked with the PRV classical gBlock, nucleic acid was
extracted and tested on the triplex assay. All of these spiked samples
were detected in the HEX and Q670 channels, and no detection was
reported in the FAM channel of the triplex assay. Similarly, seven
negative serum samples, four negative fecal swab samples and one
negative oral fluid samples were spiked with the PRV variant gBlock,
nucleic acid was extracted and tested on the triplex assay. All of these
spiked samples were detected in the FAM and Q670 channels, and no
detection was reported in the HEX channel of the triplex assay.

The triplex assay was further evaluated at the NVSL using archived oral
and nasal swabs collected from a vaccine challenge experiment conducted
in 2015 (NVSL, unpublished data). In the experiment, seven weaned
piglets were vaccinated with a USDA licensed, commercial gE/gI deleted
PRV marker vaccine; and five piglets were used as non-vaccinated
controls. All pigs were challenged with the highly virulent PRV variant
strain HeN1. A total of 116 nasal and oral swabs from this experiment
were tested on the triplex assay (24 samples each from -14, 0, 2 and
5-days post challenge and 20 samples each from 8-days post
challenge).The assay detected variant PRV starting 2 days post challenge
in all the samples. With the exception of two samples which were near
the limit of detection on the triplex assay, the results were in
agreement with the NVSL-run gB real-time PCR (Ma et al. 2008; Zanella et
al. 2012). The triplex assay did not detect any classical PRV genomes in
any of these samples tested (data not shown). As positive controls for
the triplex assay, known negative nasal and oral swab samples were
spiked with gBlocks representing PRV variant, PRV classical, and
beta-actin.

\textbf{Discussion:}

Pseudorabies (PRV) outbreaks have resulted in devastating economic
losses to affected countries, and the introduction of highly pathogenic
PRV variants from China into North America could cause severe economic
losses to the swine industry. The vaccines currently available in North
America for PRV are based on classical PRV strains and may not provide
complete protection against the highly virulent variant strains;
therefore, it is important for the veterinary diagnostic laboratories in
North America to have highly sensitive and specific diagnostic assays
that can rapidly detect and differentiate animals infected with highly
pathogenic PRV variant strains.

In September 2016, a group of researchers from China reported
development of a single-tube triplex TaqMan probe-based qPCR assay that
can differentiate classical, variant, and Bartha-K61 vaccine strains
(Meng et al., 2016). The assay used three target specific probes: one
for Bartha-K61 vaccine strain, the second for PRV classical strains, and
the third for variant PRV strains. The latter two probes were designed
based on a single nucleotide difference in the gE/gI regions of these
viruses. The assay was highly specific and evaluated using 234 field
clinical samples (all from Bartha-K61 vaccinated animals). This assay,
however, did not have a built-in internal control. PCR based assays are
sensitive to inhibitors present in extracted nucleic acids;therefore,
having an internal control in PCR-based assays is critical to confirm
the absence of PCR inhibitors in each sample. An internal control also
ensures adequate efficiency of nucleic acid extraction, which can vary
from sample-to-sample and the method of extraction used.

In this study we have developed and characterized a new single-tube
triplex real-time qPCR assay with a built-in internal control for
detection and differentiation of nucleic acid from highly virulent PRV
variant strains. The assay can be automated and can be completed within
a few hours once the samples are received in the laboratory. It has a
built-in internal control to determine assay performance. The assay was
able detect and differentiate when tested against a number of classical
PRV strains and two different variant PRV strains from China. The assay
did not detect PRV genomic DNA from commercially available, modified
live gE-deleted DIVA vaccine strains, related herpesviruses, and other
non-PRV porcine viruses tested. The assay was able to detect 0.86-1.86
copies of PRV plasmid of known DNA length and concentration and was
highly reproducible and effective on different real-time PCR machines
with comparable sensitivity on different real-time PCR platforms.

To obtain clinical samples for assessment of diagnostic performance at
the NCFAD, we collected nasal swabs every other day from pigs inoculated
with PRV JS-2012 or PRV Bristol. The assay was highly specific and
sensitive, detecting PRV nucleic acid by 4 dpi in 3-weeks old piglets
and by 6 dpi in 7-weeks old pigs. This is consistent with higher PRV
susceptibility of 3-weeks old pigs compared to 7-weeks old pigs. The
assay was further characterized using over 440 PRV negative clinical
samples collected from the U.S. and Canadian national herds as well as
positive clinical samples collected from pigs experimentally infected
with PRV classical (Bristol) and variant strains (JS-2012 and HeN1).

When compared to virus isolation, the gold standard for PRV diagnostics,
the real-time triplex assay was equally sensitive in most of the
samples, excepting two of the three trigeminal ganglia samples which
tested positive by triplex real time PCR assay but were negative by
virus isolation. This could be due to rapid establishment of latent
stage by these viruses in trigeminal ganglia in these two animals.
However, this could also indicate that the triplex real-time PCR assay
is more sensitive than the virus isolation method for PRV detection.

The triplex real-time PCR assay developed in this project could be used
as a rapid diagnostic tool for foreign animal disease detection in North
America; for routine surveillance and in epidemiological studies in
countries, like China, where both classical and variant strains are
endemic, since the assay will not detect PRV gE-deletion mutant marker
vaccines, if used.

\textbf{Acknowledgment:} This project was supported by the Swine Health
Information Centre Grant \#16-250 and the CFIA. Authors would like to
thank the animal care staff at the NCFAD-Winnipeg and the USDA National
Centers for Animal Health, Ames, Iowa, for helping with the animal
experiments. Authors would also like to thank Zoetis Inc. for providing
the PR-VAC PLUS (PRV Bucharest strain) vaccine. Additionally, the
authors thank Dr. Guang-Zhi Tong at the Shanghai Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, Shanghai, China for
providing the PRV-JS2012 strain, and Dr. En-Min Zhou at the College of
Veterinary Medicine, Northwest A \& F University, Yangling, China, Dr.
Sabrina Swenson at the National Veterinary Services Laboratories, VS,
APHIS, USDA, Ames, IA, and Melissa Goolia at the National Centre for
Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB,
Canada for their support during this project and Drs. John Copps and
Shawn Babiuk for reviewing the manuscript.

\textbf{Conflict of Interest}

None of the authors of this paper has a financial or personal
relationship with other people or organizations that could
inappropriately influence or bias the content of the paper.

\textbf{Data Availability statement}

All data related to this manuscript will be made available upon request
from the corresponding author.

\textbf{Ethics Statement (required during submission process to
Transboundary)}

The authors confirm that the ethical policies of the journal have been
adhered to. The animals used for experimental trials was approved by the
Animal Care Committee at the Canadian Science Centre for Human and
Animal Health. The guidelines from the Canadian Council for Animal Care
were observed during all procedures with animals.

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