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.
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.