Availability of a highly sensitive and specific polymerase chain reaction (PCR)-based diagnostic assay for rapid differential detection of pseudorabies virus (PRV) variants, such as those now endemic in China, is critical to prevent huge economic losses to the US and Canadian pork industries if these strains enter North America and cause an outbreak. A single-tube triplex real-time-PCR assay for differential detection of variant strains of PRV has been developed and evaluated in a project funded by the Swine Health Information Center (SHIC) and Canadian Food Inspection Agency (CFIA). 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 or for surveillance and in epidemiological studies in countries, like China, where both classical and variant strains are endemic. The assay is also able to differentiate wild-type PRV from the gE-deletion PRV mutant marker vaccines.
In response to questions posed by the National Pork Board prior to 2015, the National Veterinary Service Lab tested the ability of PCR tests available at that time in the US to detect the Chinese PRV high pathogenic variant. They found the US PCRs can detect it if it gets to this country. Now, this new triplex enhances that capability by being able to differentiate the Chinese PRV from the classical strains of PRV as well as from the vaccine strains of PRV.
The newly developed assay targets the intergenic region between the US2 and US6 genes in the PRV genome. It is highly sensitive and specific and 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.
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.
PRV causes Aujeszky’s disease or pseudorabies (PR), 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.
As the world deals with the COVID-19 pandemic, SHIC continues to focus efforts on prevention, preparedness, and response to novel and emerging swine disease for the benefit of US swine health. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. SHIC is funded by America’s pork producers to fulfill its mission to protect and enhance the health of the US swine herd. For more information, visit https://www.swinehealth.org or contact Dr. Sundberg at [email protected].