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A single-tube triplex real-time quantitative PCR assay for differential detection of highly virulent Chinese strains of pseudorabies virus
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  • Orlando Perez,
  • Mathieu Pinette,
  • Jianfa Bai,
  • Lalitha Peddireddi,
  • John Schiltz ,
  • Karthik Shanmuganatham,
  • Rachel Tell,
  • Alfonso Clavijo,
  • Aruna Ambagala
Orlando Perez
Canadian Food Inspection Agency
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Mathieu Pinette
Canadian Food Inspection Agency
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Jianfa Bai
Kansas State University
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Lalitha Peddireddi
Kansas State Veterinary Diagnostic Laboratory, KansasState University
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John Schiltz
USDA National Veterinary Services Laboratories
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Karthik Shanmuganatham
USDA National Veterinary Services Laboratories
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Rachel Tell
USDA National Veterinary Services Laboratories
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Alfonso Clavijo
USDA
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Aruna Ambagala
Canadian Food Inspection Agency
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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.