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