Introduction:
Pseudorabies virus (PRV), or suid herpesvirus-1, is a double stranded
DNA virus of the genus Varicellovirus , subfamilyAlphaherpesvirinae , and family 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 naïve 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.