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.