Diagnostic Genetic Techniques for PRRS Viruses

Polymerase chain reaction (PCR) is a lab procedure that artifically copies the genetic material of the PRRS virus. PCR copies one tiny strand of PRRS virus RNA into millions of strands that are exactly alike. Once a large number of copies have been made, other diagnostic procedures can be performed.

Sequencing is one such procedure that uses the PCR product. Sequencing determines the order of the genetic base units that make up the RNA strand of the PRRS virus. Knowledge of the order of the base genetic units allows identification of individual PRRS viruses. Each PRRS virus type has a unique order of base units.

Differentiation between multiple PRRS virus types can be important in a swine production system, as different types of virus may not be combated by current system immunity. The symptoms related to PRRS exposure are dependent on whether or not the immunity of the pigs in a system can recognize a virus type. Three methods are often used in PRRS diagnostics to differentiate types of PRRS virus sequences. The methods are restriction fragment length polymorphism (RFLP) (Larson, 1994), sequence comparison (Felsenstein, 1993 and Higgins, 1994), and phylogenetic analysis (Felsenstein, 1993 and Higgins, 1994).

RFLP cut patterns are easy to use to qualify different PRRS viruses. RFLP divides or "slices" a known genetic strand at every point a specific arrangement of base units occurs. RFLP cuts the genetic strand into many pieces. Three segmental pieces are identified after cutting and the quantities of the three pieces are counted. As an example, a 1,4,2 RFLP cut pattern is a PRRS virus that after cutting accumulates only one segmental piece of the first kind, four pieces of the second, and two pieces of the third. RFLP is not very sensitive for differentiating PRRS virus types as many nonidentical viruses share the same cut patterns. Uncommon cut patterns and vaccine patterns are the most informative results found in most systems.

Sequence comparison allows the determination of whether a PRRS virus is the same or different than another PRRS virus. Comparison is done by physically overlaying the base unit sequence of one PRRS virus on the sequence of another and counting the differences in base units between the two sequences. Identical viruses have 15 or fewer differences. Viruses that are genetically dissimilar have 40 or more differences. The guidelines for tallied differences are applicable only to genetic sequences of PRRS viruses derived from a specific region of the virus known as open reading frame 5 (ORF 5) and open reading frame 6 (ORF 6). This region of the PRRS virus is the most common region sequenced by veterinary diagnostic laboratories.

A phylogenetic tree is a "family tree" that compares the sequences from a multiple of different PRRS virus isolates. The vertical order of tree places similar viruses close together. Horizontal lines connect various viruses. Similar viruses and "viral families" are connected with shorter horizontal lines. The longer the lines extend to the left to connect two viral families, the less related are the two families. Horizontal line length is from a computer determination of clustal residue.

Case Report

Following is a case report that compares the types of PRRS viruses found in two systems owned by a swine production company. The first system, System 1, is comprised of about 55,000 sows on 12 sow farms. The other system, System 2, is comprised of about 40,000 sows on 13 sow farms.

Both systems use 3-site production. Both systems use some 2500-head and 5000-head sow units. The same genetic company is used to source the two systems with animals of the same genetic background. Feed rations are the same, but mills are different. Management personnel and programs are different between the two systems.

Over 100 PRRS virus specimens were isolated across the two systems and copied by PCR. The PCR product strands were then sequenced and RFLP cut patterns identified. Many of the sequences were organized into a phylogenetic tree. The phylogenetic tree distinguished six PRRS virus families. The families stratified the viruses by system. Viral families identified in one system were not found in both.

The distribution of viral family types by "system" or geography has been shown with viruses other than PRRS. The stratification AIDS viruses by a phylogenetic tree depicts nine families residing separately in different parts of the world (Sanchez, 1995). Human astroviruses have seven distinct families with some geographic separation (Noel, 1995). Rabbit hemorrhagic disease virus was shown to have four distinct families separated across time in France (Le Gall, 1998).

It was concluded that System 1 was infected with fewer types of PRRS viruses than System 2. System 2 was estimated to have at least four times as much variation in the genetic content of its PRRS viruses than did System 1. System 1 has retained one dominant family of PRRS virus for over two yeas. The multipliers, commercial sow farms, nurseries, and finishers share the same virus. The stability of the dominant family in System 2 contradicts the theory that PRRS virus recombination lead to instability over time. The following table describes the variation noted in PRRS viruses comparing the two systems.

The annual sow production statistics of the two systems are similar. The incidence of acute PRRS in sow farms was more common and more severe in System 2, especially during the last two quarters. The sow farm production data of the last 6 months has been affected more than annual data indicates. Annual nursery mortality is 48% higher in System 2 as compared to System 1. The same trend is reflected in finishing.

Both systems adopted the same approach to PRRS management two years previously. Implementation of the program was more disciplined in System 1 as compared to System 2. PRRS control was implemented as a three-prong approach. First priority was to avoid introducing new PRRS virus types through outside seed stock sources. System 1 and System 2 shared the same genetic sources and multiplication. Initially, only PRRS negative animals were entered into the company. Later, genetic material was introduced only in the form of semen.

The second priority was to acclimatize replacement gilts and boars to the PRRS viruses of commercial sow farms before they were entered into each commercial herd. System 1 and System 2 had similar acclimation procedures, although not the same.

The third priority was to consistently source replacement gilts and boars from one specific multiplication farm to supply commercial sow farms in a pyramid. System 1 was disciplined and flowed replacement gilts and boars only from the assigned multiplier farms of each pyramid. System 2 used its multiplier farm, but allowed violations.

System 2 had one multiplier farm and it produced inadequate quantities of off spring to supply the animals needed to start new herds within the system. Two new sow farms (10,000 sows) were stocked from five different multipliers outside the company. In addition, the System 2 multiplier could not provide replacements for the two new farms. Replacements were received for over a year from one multiplier outside the company. All pigs from System 2 were entered into the same nurseries.

The System 2 multiplier output was also inadequate to meet the needs of existing commercial sow farms. A plan was devised that eventually introduced PRRS positive replacement gilts from a source outside the company into every System 2 commercial sow farm. A PRRS virus was found December '97 in the isolated outside replacement gilts before they were entered into the commercial farms. The same virus was isolated during clinical PRRS outbreaks on commercial farms in March '98 after the gilts were entered. The same virus was again found in January '99 in outbreaks occurring in System 2 commercial farms. The phylogenetic tree indicates the close relationship of the isolates.

A similar tracing of viral origin through a phylogenetic tree was used by the Danes to document the introduction of a new strain of PRRS into Danish swine populations. The Danes showed that the introduction of a vaccine strain developed into related new infective strains (Madsen, 1998).

Changing to a larger multiplier farm was the final method chosen to increase the quantity of replacement animals available to System 2. The new is farm was twice as large, but is located in System 1. The endemic virus in the new multiplier farm is genetically different than the viruses found in the rest of System 2. The change of multipliers is recent and the phylogenetic tree does not indicate an effect of this action yet.

Take-Home Message

The report suggests that more clinical losses may occur due to PRRS when a multiplicity of PRRS virus types is present within a system. This report also suggests that sourcing PRRS positive replacement gilts and boars outside the pyramid multiplier may be a great risk for increasing the amount of PRRS associated disease losses. "Creative replacement flows" that offer quick answers to production bottle necks are dangerous to system PRRS stability if designed replacement flow is voided. The perceived threat of viral recombination to system PRRS stability might be exaggerated.

References

Felsenstein J., 1993, PHYLIP (phylogeny interference package): version 3.5c. Department of Genetics, University of Washington.

Higgins D.G., 1994, CLUSTAL V: Multiple alignment of DNA and protein sequences. Methods in Molecular Biology. 25: 307 - 318.

Larson T.J. and Bender P.K., PC/GENE: Restriction enzyme analysis. Methods in Molecular Biology. 24: 267 - 274.

Le Gall G., Arnauld C., Boilletot E., Morisse J., and Rasschaert D., 1998, Molecular epidemiology of rabbit hemorrhagic disease virus outbreaks in France during 1988 to 1995. Journal of General Virology. 79: 11 - 16.

Madsen K.G., Hansen C.M., Madsen E.S., Strandbygaard A.B., and Sorensen K.J., 1998, Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds. Archives of Virology. 143: 1683 - 1700.

Noel J.S., Lee T.W., Kurtz J.B., Glass R.I., and Monroe S.S., 1995, Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing. Journal of Clinical Microbiology. 43: 797 - 801.

Sanchez S.P., Dopazo J., Olivares I., Martin M.J., and Lopez C.G., 1995, Primary genetic characterization of HIV-1 isolates from WHO-sponsored vaccine evaluation sites by the RNase-A mismatch method. Virus Research. 39: 251 - 259.