What are porcine reproductive and respiratory syndrome RFLPs?

How do we use them and should there be a new naming scheme?

April 12, 2022

5 Min Read
National Pork Board

Several different strategies have been used to classify porcine reproductive and respiratory syndrome-type-2 viruses into (hopefully) epidemiologically meaningful groups. Here we discuss RFLPs (Restriction Fragment Length Polymorphisms), one of the most commonly used ways of classifying this virus, highlighting how this classification works and its limitations.

RFLP classification for PRRSV was originally proposed in 1998 as a way of differentiating wild (field) strains from vaccine strains (1) at a point of time when sequencing was challenging and expensive. Originally, three restriction enzymes (MluI, HincII and SacII) were used to fragment or cut the PRRSV ORF5 gene, each binding to a specific pattern within the RNA sequence that may occur at different locations across the ORF5 gene. The location (or locations) where each enzyme binds was determined through a laboratory technique called gel electrophoresis. Various patterns of where these enzymes bind and cut the ORF5 gene were denoted as RFLP cut patterns. Today, viral RNA does not need to be exposed to enzymes or run through gel electrophoresis, as RFLPs can be determined computationally from ORF5 sequences.

While originally created to differentiate wild and vaccine strains, the industry expanded the use of RFLP types to further classify the virus into subtypes and help address additional epidemiological questions. For example, are two wild-type viruses the same? Do they behave in similar ways? Are they related evolutionarily? Those questions began being addressed by looking at the RFLP pattern of the virus, but the answers were at best unclear.

One of the issues with RFLP typing is that it does not necessarily tracks genetic relationship. RFLPs have been shown to change in as few as 10 animal passages (3), and a single mutation (depending on where it occurs within ORF5) has the potential to change the RFLP type. This is referred to as "RFLP instability." Additionally, distantly related PRRS viruses regularly share the same RFLP type (see Supplementary Table 1 of Paploski 2021) (4). These factors make the immunological meaning of RFLP types unclear, partly because it is not always clear whether viruses that share the same RFLP type are homologous and whether viruses with different RFLP types are truly heterologous. 

So how are RFLPs used in the daily routine of a farm? The first use is to help differentiate wild-type PRRS from strains utilized in some modified live vaccines. Some vaccines in the market have a particular RFLP pattern that is practically exclusive to the vaccine. This is not true to all vaccines commercially available.

Another use of RFLPs is for the characterization of a virus, for example, during an outbreak. Because RFLPs are so easy to determine after viral sequencing has been performed, they can be used as a "quick and dirty" way to characterize which virus is involved in a given outbreak, and potentially determine if the detected virus is the same or different from other PRRSV viruses circulating in the farm or system. However, this characterization is not always extremely meaningful or informative, given the caveats of RFLP classification discussed above. 

One way to minimize the limitations of RFLP is to use RFLPs in combination with other classification methods, such as phylogenetic lineages, to allow for a better identification of meaningful viral groups that are genetically related. A recent example of this is the newly emerged Lineage 1C RFLP 1-4-4 variant (2). When first identified, this virus was initially described as a RFLP 1-4-4. However, only 33% of the RFLP 1-4-4 viruses currently circulating in the United States were associated with the viral cluster that caused this outbreak, creating confusion.

Alternatively, according to lineage-based classification, this novel variant is a member of sub-lineage 1C (L1C). However, L1C is a very large family of viruses and only 42% of L1C viruses that are currently circulating are associated with this outbreak. By combining both classification methods, 81% of all currently circulating L1C-1-4-4 viruses were associated with this outbreak. While still not perfect, combining both methods allows a quick yet more accurate determination of whether a given virus is part of the recent L1C-1-4-4 outbreak.

Given modern advances in sequencing technology and widespread use of ORF5 sequences, it is becoming increasingly apparent that using RFLP-types to refer to PRRSV  type-2 viruses is both outdated and, more importantly, can potentially lead to misleading ov even erroneous conclusions.  While the shortcomings of RFLP-types have long been recognized, no alternatives have yet been proposed.  Lineages and sub-lineages attempt to provide more biologically meaningful classification for PRRS viruses, but do not have the level of granularity often required for on-farm management of PRRSV.

We want to know — what do you think about the current PRRS nomenclature? 

Results of this five-question survey (link here) aim to assess how PRRSV sequence data and related nomenclature are being used in the industry, and identify attributes of an ideal naming system that would meet the needs of swine practitioners to track PRRS at the farm, system, regional and national scales. This survey will provide information for initiatives to update PRRS nomenclature.

1. Wesley R, Mengeling W, Lager K, Clouser D, Landgraf J, Frey M. Differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from North American field strains by restriction fragment length polymorphism analysis of ORF 5. J Vet Diagn Invest. 1998;10(2):140–4.
2. Kikuti M, Paploski IAD, Pamornchainavakul N, Picasso-Risso C, Schwartz M, Yeske P, et al. Emergence of a New Lineage 1C Variant of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States    [Internet]. Vol. 8, Frontiers in Veterinary Science  . 2021. Available from: https://www.frontiersin.org/article/10.3389/fvets.2021.752938
3. Cha S, Chang C, Yoon K. Instability of the Restriction Fragment Length Polymorphism Pattern of Open Reading Frame 5 of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Pig-to-Pig Passages. J Clin Microbiol. 2004;42(10):4462–7.
4. Paploski IAD, Pamornchainavakul N, Makau DN, Rovira A, Corzo CA, Schroeder DC, et al. Phylogenetic Structure and Sequential Dominance of Sub-Lineages of PRRSV Type-2 Lineage 1 in the United States. Vaccines [Internet]. 2021 Jun 5;9(6):608. Available from: https://www.mdpi.com/2076-393X/9/6/608

Source: Igor Paploski and Kimberly VanderWaal, who are solely responsible for the information provided, and wholly owns the information. Informa Business Media and all its subsidiaries are not responsible for any of the content contained in this information asset.

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