Molecular diagnostic tools help distinguish wild-type and vaccine strains of PRRSV, By Phillip Gauger, DVM,

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge the US swine 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 paper.1

Fortunately, multiple molecular diagnostic assays are available for detecting, sequencing and characterizing PRRSV, said Gauger, who is a diagnostic pathologist with the Iowa State University Veterinary Diagnostic Laboratory and leader of the molecular diagnostics section.

Challenges with PRRSV

“One particular challenge facing the industry has evolved through the use of modified-live PRRSV (MLV-PRRSV) vaccines and the inability of diagnostic tests to distinguish between the detection of wild-type PRRSV (WT-PRRSV) and/or vaccine-like 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 genetically evolve through point mutations in the genome; and may experience recombination with WT-PRRSV.”

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

New testing options available

New molecular testing methods have been developed to not only detect PRRSV in a specimen but also characterize the presence of different PRRSV strains in a production system. These methods attempt to distinguish between detection of a 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 between the presence of wild-type and vaccine strains of PRRSV.

“CLAMP is a term — not an acronym — to suggest what the assay will do during its performance,” Gauger said. “Thus, it clamps down on the homologous sequence of a MLV-PRRSV and prevents amplification and sequencing of that gene. CLAMP is the bridged nucleic acid designed to bind tightly to the homologous target, when present, and ultimately allows preferential sequencing of a WT-PRRSV.”

Numerous molecular diagnostic assays are presently available to detect PRRSV in swine diagnostic specimens, Gauger said, but each one has certain benefits and limitations (see box).

Future promising

Next-generation sequencing offers a range of processes that can provide the whole-genome sequence of a specific virus, Gauger explained. It can also detect the presence of novel viruses or allow for deep sequencing of the microbiome.

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

“Although no molecular diagnostic test is perfect, these tests and combinations of different assays improve the probability of detecting and characterizing a strain or multiple strains of PRRSV present in a group of pigs,” Gauger added.

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

Existing Diagnostic Tools

PRRSV-screening real-time polymerase chain reaction (PCR) assay: These tests are highly 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 capable of differentiating different genetic strains of the virus, Gauger said. If a PRRSV-screening PCR assay is positive, additional testing may be needed.

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

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

PRRSV CLAMP sequencing assays: This assay is used when both MLV-PRRSV and WT-PRRSV are suspected in a population of pigs. The CLAMP process utilizes a bridged nucleic acid as described earlier to prevent amplification and sequencing of MLV-PRRSV, allowing preferential sequencing and detection of WT-PRRSV, if present. It can detect WT-PRRSV sequences of lower concentration in vaccinated pigs and helps reduce the risk of transmitting a WT-PRRSV that could remain undetected without the more advanced molecular tools currently available, Gauger said.



1 Gauger P, Harmon K. Iowa State University Veterinary Diagnostic Laboratory, Department of Veterinary Diagnostic and Production Animal Medicine, Ames, Iowa. PRRS CLAMP: molecular diagnostic tools to distinguish wild-type and vaccine strains of PRRSV. Presented at Am Assoc Swine Vet annual meeting, March 2021.