New Test Differentiates Chinese PRV from Classic PRV in Pigs

( National Pork Board and the Pork Checkoff )

An endemic strain of pseudorabies virus (PRV) is circulating through China’s pig herd and causing major economic losses to pigs in all stages of growth. Although this virus is currently not in the U.S. commercial swine herd, it continues to be a disease of top interest because of its potential to cause widespread economic loss and devastation.

“Pseudorabies is one of the top interests in the swine disease matrix list,” says Paul Sundberg, Swine Health Information Center (SHIC) executive director. “Since 2011, highly virulent PRV strains that are genetically different from the classic PRV strains surfaced in pig herds in China. We can't forget the lessons of PEDV. All it takes is a one-time event to introduce a pathogen into the country.”

Although it’s not considered an “official” foreign animal disease, Sundberg says it is just as important to keep out as African swine fever (ASF), classical swine fever and foot-and-mouth disease. And that’s where SHIC comes in – ensuring the country has adequate diagnostics for emerging disease detection. 

SHIC has been trying to learn as much as it can about the highly pathogenic PRV strain that is causing so much trouble in China. 

“What we're trying to do is ensure that we're being proactive in covering as many bases as we can,” he adds. “We need to learn about that virus while it's over there in preparation for it getting here. Our first question was simple. Can our current polymerase chain reaction (PCR) tests detect the highly pathogenic pseudorabies that is circulating in China? The answer ended up being yes, those PCR tests can detect it.”

But Sundberg says they needed to take it another step further. Even if they could detect it, would they be able to differentiate what strain of PRV they were dealing with? 

New Assay Detects Variant Strains
SHIC teamed up with the Canadian Food Inspection Agency (CFIA) to develop a single-tube triplex real-time-PCR assay for differential detection of variant strains of PRV. The triplex real-time PCR assay developed can 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, Sundberg says.

The newly developed assay targets the intergenic region between the US2 and US6 genes in the PRV genome, SHIC explains in a release. The test is highly sensitive and specific and did not detect other non-target viruses. 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, the release says. 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, with the exception of two of the three trigeminal ganglia samples which tested positive by triplex real time PCR assay but were negative by virus isolation. SHIC reports this could be due to rapid establishment of the 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.

What’s Next
In order to protect the U.S. swine industry, Sundberg says these PCR tests must be implemented through the National Animal Health and Laboratory Network (NAHLN) and made available to all diagnostic labs as an official test. 

“It's a validated test with a lot of power to it because it was developed in a government laboratory with our partners in Canada,” Sundberg says. “Now it has to be distributed in order to make it usable.”

If an outbreak of pseudorabies were to occur in the U.S. or Canada, Sundberg says the tests can be deployed very quickly through an emergency distribution. He hopes the North American pork industry doesn’t have to wait for an outbreak but can get the tests deployed to laboratories ahead of time. Currently, only a couple of diagnostic labs in the NAHLN system have a pseudorabies PCR, he adds.

“If a diagnostician takes a look at a sample and it looks like pseudorabies, they have to send the sample to the National Veterinary Services Laboratory (NVSL) in Ames, Iowa, and request it be tested for pseudorabies,” Sundberg explains. “That's a strong system, but it's not a timely one. It may be delayed because there's got to be that transfer of the samples.” 

Sundberg believes this PCR test is an important step forward and now is the time to fully take advantage of the improvements that have been made to the tests. 

“This test itself will benefit the North American pork industries. It can help us quickly differentiate sources of viruses and detect pseudorabies incursion. Our hope is that we can make sure the test is quickly usable within the diagnostic labs themselves,” he says.

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