Molecular diagnostic tools help distinguish wild-type and vaccine strains of PRRSV


The porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge the US pork industry. The virus has become more difficult to control despite aggressive biosecurity methods, multiple commercial vaccines, live virus inoculation methods and the implementation of vaccination protocols, said Phillip Gauger, DVM, PhD, in a recent research article.1

Fortunately, there are several molecular diagnostic tests available to detect, sequence, and characterize PRRSV, said Gauger, a diagnostic pathologist in the Iowa State University Veterinary Diagnostic Laboratory and head of the molecular diagnostics section.

Challenges with SDRPV

“A particular challenge facing the industry has evolved through the use of modified live PRRSV vaccines (MLV-PRRSV) and the inability of diagnostic tests to distinguish between detection of wild-type PRRSV (WT -PRRSV) and / or vaccine type. strains of the virus, ”Gauger said.

“Vaccine-like MLV-PRRSV can replicate in vaccinated animals for long periods of time; is capable of transmission to unvaccinated pigs; can evolve genetically thanks to point mutations in the genome; and can undergo recombination with WT-PRRSV.

These factors can complicate the overall epidemiology or genetic ecology of PRRS, and several tests or sequencing tests may be required to distinguish between the presence of MLV-PRRSV or WT-PRRSV when mixed infections are suspected, a Gauger said.

New test options available

New molecular test methods have been developed to not only detect PRRSV in a sample, but also characterize the presence of different strains of PRRSV in a production system. These methods attempt to distinguish between detection of an MLV-PRRSV, WT-PRRSV or multiple strains of the virus, Gauger explained.

PRRS CLAMP refers to a molecular diagnostic sequencing tool that can help distinguish the presence of wild-type and vaccine strains of PRRSV.

“CLAMP is a term – not an acronym – to suggest what the test will do while it is running,” Gauger said. “Thus, it blocks the homologous sequence of an MLV-PRRSV and prevents the amplification and sequencing of this gene. CLAMP is the bridged nucleic acid designed to bind tightly to the homologous target, when present, and ultimately enables preferential sequencing of a WT-PRRSV.

There are many molecular diagnostic tests currently available to detect PRRS in pig diagnostic samples, Gauger said, but each has certain advantages and limitations (see box).

Promising future

Next-generation sequencing offers a range of processes that can provide the entire genome sequence of a specific virus, Gauger explained. It can also detect the presence of new viruses or allow in-depth sequencing of the microbiome.

“For routine diagnostic purposes, whole genome sequencing of PRRSV may be an appropriate option to answer a diagnostic question or monitor the epidemiology of a PRRSV circulating in a production system,” he said. declared.

“While no molecular diagnostic test is perfect, these tests and combinations of different tests improve the likelihood of detecting and characterizing one or more strains of PRRSV present in a group of pigs,” Gauger added.

“They also help reduce the risks associated with moving pigs unknowingly infected with WT-PRRSV, especially when MLV-PRRSV is present in the same population. “

Existing diagnostic tools

Real-time polymerase chain reaction (PCR) assay for PRRSV screening: These tests are very sensitive and can detect low concentrations of PRRSV and all strains. They are highly specific and multiplexed for PRRSV type 1 or type 2 species, but are not designed for further characterization of the virus nucleic acid and are not able to differentiate between different genetic strains of the virus, a Gauger said. If a PCR test for PRRSV is positive, additional testing may be necessary.

Sequencing of the PRRSV ORF5 gene: This test is a molecular diagnostic test based on the amplification of the ORF5 gene followed by the selective determination of the nucleotide sequence within the gene. “Diagnostic reports with the ORF5 sequence data will also include a comparison of nucleotide identity with the MLV-PRRSV vaccine strains,” Gauger said.

Real-time PCR by reverse transcription of the PRRSV vaccine type: “This assay can detect the presence of MLV-PRRSV and is suggested for use in PRRSV monitoring protocols in combination with other diagnostic assays (PRRSV-screen PCR and / or sequencing),” Gauger said. “Or, it can be used when the history, clinical impressions and use of the vaccine are well known to justify the use of this test,” Gauger said.

PRRSV CLAMP Sequencing Tests: This test is used when MLV-PRRSV and WT-PRRSV are suspected in a pig population. The CLAMP method uses a bridged nucleic acid as described above to prevent amplification and sequencing of MLV-PRRSV, allowing preferential sequencing and detection of WT-PRRSV, if present. It can detect lower concentration WT-PRRSV sequences in vaccinated pigs and helps reduce the risk of transmitting WT-PRRSV that could go undetected without the most advanced molecular tools currently available, Gauger said.

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