Commentary

Non refereed

The potential role of genetic recombination in the evolution
of new strains of porcine reproductive and respiratory syndrome virus (PRRSV)

William L. Mengeling, DVM, PhD, Diplomate ACVM

Collaborative Professor, Department of Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa. Tel: 515-292-7060; E-mail: bbmengeling@aol.com.

Mengeling WL. The potential role of genetic recombination in the evolution of new strains of porcine reproductive and respiratory syndrome virus (PRRSV). J Swine Health Prod. 2002;10(6):273-275.

Over the past several years, there has been a great deal of interest in the idea that "new" strains of porcine reproductive and respiratory syndrome virus (PRRSV) often emerge as a result of genetic recombination among existing strains. Although we may never definitively know the relative contribution of recombination in creating PRRSV diversity, the possibility that it plays an important role is an inviting concept for several reasons.

First, recombination would help explain the appreciable differences in genetic sequence among various isolates of PRRSV. In some cases, these differences are so great that one might question whether they are entirely the result of point mutations.¹ Of course, mutation must also be involved to a considerable extent, because without prior mutational changes, ie, if all existing PRRSV had an identical sequence, recombination might be a moot point.

Second, recombination would help explain events such as the sudden appearance of an unusually severe form of PRRS (often referred to as "atypical" or "acute" PRRS) in the midwest in the fall of 1996.² Isolates from at least some of these epidemics were seemingly more virulent than strains that had been isolated before that time.³ Although it's possible that one or more point mutations caused this perceived increase in virulence, recombination seems a likely candidate because of its greater potential for immediate phenotypic change. As an aside, a common restriction fragment length polymorphism (RFLP) pattern, namely, 1-4-2,^4,5 among most isolates from atypical PRRS cases recognized at that time, suggested a common ancestor that predominated and spread widely in the swine population (the importance of predominance in regard to the potential clinical impact of recombination will be discussed in more detail below). If so, the means by which they spread so rapidly through the swine population in the United States and Canada is still unclear.

Third, at least four reports emphasized the potential involvement of recombination in the evolution of "new" strains. These included the documentation of strong statistical evidence for intragenic recombination;¹ the identification of recombinants following co-infection of cell cultures with two attenuated strains of the North American type of PRRSV;⁶ the identification of recombinants following co-infection of cell culture with two European strains of PRRSV;⁷ and the evolution and subsequent predominance of recombinants in pigs simultaneously exposed to five attenuated strains of PRRSV.⁸ By sequencing the entire genome of several of the recombinants isolated in the latter study, it was discovered that some comprised segments of as many as four of the parental strains (unpublished data, 2001).

From the collective information provided by these reports one might theorize that recombination is a common means by which PRRSV changes in the field. Unfortunately, the facts at hand tell only part of the story. We might assume that genetic recombination among strains of PRRSV is a common event under natural conditions. Of course, this issue is debatable for a number of reasons, the first of which is that most pigs are probably infected with only one strain at a time unless vaccinated in the face of, or shortly before, an epidemic. However, even if we knew the actual incidence of recombination, we would still be unsure of how often such recombinants predominate in their host. Probably most have an ephemeral existence. This point is emphasized by the fact that recombinants created by simultaneous infection of cell cultures with two strains of PRRSV apparently disappeared from the mixture during the course of additional cell culture passages.⁶

It might be argued that the study with attenuated strains of PRRSV, wherein recombinants did, in fact, predominate in pigs,⁸ would suggest otherwise. But keep in mind the artificiality of the experimental design in regard to field conditions. How often are pigs simultaneously exposed to two strains of attenuated PRRSV, much less five? In addition, the experimental design was purposely biased toward predominance by using attenuated strains - an issue that will become clearer from subsequent state-ments.

On the basis of potential clinical and economic impact, PRRSV recombination can be sorted into three general categories: recombination between or among virulent strains; recombination between or among attenuated strains; and recombination between or among virulent and attenuated strains. Each category can be further defined by the likelihood of the actual event of recombination and the likelihood that a recombinant will survive and eventually predominate (ie, out-replicate its parents so as to be the primary or only strain shed from the infected pig).

Recombination between or among virulent strains

We may reasonably assume that there will be strain recombination if a pig is infected with two or more virulent strains of PRRSV at or about the same time. One might even speculate that it would be common if such strains had a similar rate of replication and thus coexisted for weeks or months. Surely during this long interval some cells would be co-infected, thus setting the stage for recombination. However, the emergence of a recombinant as the predominant strain is less common. Fortunately, there is an upper limit to the rate of PRRSV replication (this applies to other viruses as well) and the virulent parents are likely to be at or near that limit. Consequently, there is little opportunity for the recombinant to enhance its minority status unless other factors come into play. And so it would probably never see the "light of day". On the other hand, if for some reason recombination did result in a strain with even a slight replication advantage, and thus probably an added level of virulence, it might eventually become established in the swine population with serious consequences. Isolates from epidemics of atypical PRRS were previously mentioned as such a possibility. In any case, the purposeful exposure of swine to two or more virulent strains of PRRSV should probably be discouraged. For example, if virulent PRRSV is used to establish immunity in gilts prior to their entry into the breeding herd, it would seem advisable to make sure the inoculum or contact exposure comprised a single strain.

Recombination between or among attenuated strains

Although experimental studies have established that attenuated strains of PRRSV can recombine in cell cultures⁶ and in pigs,⁸ and that recombinants of attenuated parents can predominate in pigs,⁸ there remains a question of whether such recombinants are likely to present a clinical problem (at least relative to fully virulent strains). To address this question in part, let's again assume that predominance is a consequence of replication advantage, and that there is a direct correlation between the rate of replication and virulence. It follows that any recombinant (of attenuated parents) that subsequently predominates in its host is likely to be less attenuated (or more virulent, depending on one's perspective) than its parent strains. Note that in this case, the parent strains, by virtue of attenuation, replicate in pigs at a rate appreciably less than that of virulent strains, so there is room for the recombinant to move toward the upper limit of replication rate. On the other hand, there is no reason to believe that the recombinant of attenuated parents would have gained a replication rate and level of virulence equal to a fully virulent field strain. Support for this idea is provided by a recent finding that when a recombinant of attenuated parents was tested in pigs, it was found to be somewhat more virulent than its parents, but markedly less virulent than a virulent field strain tested under similar conditions (unpublished data, 2001). Nevertheless, the probability that attenuated-strain recombinants are less attenuated than their parents argues against the use of multi-strain PRRSV vaccines - despite the potential for such vaccines to stimulate a broader spectrum of immunity.^9,10

Recombination between or among virulent and attenuated strains

Probably dual (strain) infection occurs most frequently when pigs are vaccinated either at a time when they are already infected with virulent virus, or when (or shortly before) they are exposed to virulent virus. Fortunately, recombination between virulent and attenuated strains of PRRSV seems less likely than recombination between strains of similar virulence. The reason is that virulent strains quickly predominate, and, as a consequence, dual infection is relatively short-lived. For example, when pigs were simultaneously exposed to as many as 400,000,000 median cell culture infectious doses (CCID₅₀) of attenuated (vaccine) virus and only 40 CCID₅₀ of virulent virus, only virulent virus was isolated from their blood at both 7 and 14 days later.¹¹ This result becomes clearer when we consider that, in pigs, typically tenfold or more virulent virus than attenuated virus is produced during their respective replication cycles. In addition, the replication cycle for PRRSV is relatively short, namely about 8 hours in cell cultures (unpublished data, 1994) and probably about the same in pigs. With this in mind, it is obvious that it wouldn't take many days for virulent virus to reach majority status despite any initial handi-cap.There is additional comfort in the fact that even if recombination occurred, it is unlikely that the recombinant would predominate. That is, why would a recombinant, derived in part from an attenuated strain, out-replicate its virulent parent? And if it did, why would it be of appreciably greater virulence than its virulent parent? Nevertheless, the probable frequency at which pigs are infected with both attenuated (vaccine) and virulent strains makes recombination a possibility for consideration. Perhaps its greatest impact would be to present a diagnostic dilemma. Imagine a recombinant comprising mostly virulent virus other than for segments typically used for strain identification, namely open reading frames 5, or 5 and 6. Although this type of construct seems improbable, it can't be excluded. And its mere possibility suggests that a suspected vaccine derivative should be fully sequenced or examined in more than one open reading frame by RFLP analysis¹² before assuming its ancestry - particularly if the conclusion is going to have any relevance in regard to PRRS control.

Although emphasis has been placed on a direct correlation between a genetically controlled rate of replication and predominance, there are other means by which a recombinant could gain a competitive advantage. One possibility is immune selection, whereby humoral immunity or cellular immunity or both might be more effective against the antigenic mosaic present for the longest time, namely, the parent strains. Although we can't exclude the potential role of selective immunity, the pig's relatively feeble neutralizing antibody response to PRRSV makes this less likely for PRRSV than for many other more "immunogenic" viruses. Another possible selective factor is receptor affinity, whereby the recombinant might be better able to attach or adhere (or both) to susceptible cell receptors - which in turn would likely promote a greater incidence of infection. We know, for example, that PRRSV propagated a large number of times in cell culture, namely, 251 times in MARC-145 (monkey kidney) cells, may be poorly infectious for pigs until it has been back-passaged in pigs at least one time (unpublished data, 2000). We theorize that this reflects a change in receptor affinity, induced, at least in part, by propagating the virus in non-porcine cells with similar, but not identical, receptors.

Whatever the prevalence and circumstances of recombination and predominance, there is yet another hurdle to clear if a recombinant is to be of any appreciable clinical and economic relevance. Namely, it must spread widely and become established in the swine population. Consequently, any on-site strategy that minimizes the spread of pathogens in general will also have an impact on the emergence of "new" strains of PRRSV. This emphasizes the importance of herd and site biosecurity in regard to exit as well as entrance of pathogens, and it emphasizes the need for an industry-wide PRRSV control program.

So far, the focus has been strictly on inter-strain recombination. However, it's conceivable that intra-strain recombination¹³ during replication of PRRSV is also involved in promoting and accelerating genetic change - albeit at a much slower pace. Imagine a cell being infected with a single virus particle, but then during replication, numerous permutations of the original genome arising as a result of point mutations. We might speculate that recombination among these putative quasi-species¹⁴ during the remainder of the replication cycle in the same cell would be much more common than recombination among different strains because, with the exception of the mutation sites, the remaining sequences would be identical,⁷ and because every infected cell would be a cauldron for recombination. That is, the requirement for a cell to be co-infected with two different strains of PRRSV would be circumvented. Before leaving this topic of inter- and intra-strain recombination, it is important to emphasize that there is no universally accepted definition of "strain" for PRRSV. The sequence diversity among most isolates of PRRSV has resulted in the relatively promiscuous use of the term "strain," even though there often are no discernible phenotypic differences. That doesn't mean that there are none: it may be simply that, in most cases, we don't as yet have the tools for their identification.

Summary

Genetic recombination will continue to be a topic of special interest in regard to the epidemiology and control of PRRS. Presumably, its prevalence in a particular pig depends on infection with strains of similar virulence (which in turn tends to promote co-infection over a long period of time) and a similar sequence over at least some segments of the genome. From a practical perspective, genetic recombination isn't the whole story. A recombinant must then clear the additional hurdles of predominance and spread to be of appreciable clinical and economic relevance. The fact that PRRSV typically persists and replicates in an infected pig for 6 or more weeks makes it a better candidate than most other porcine viruses for genetic diversity and evolution via recombination. The potential for recombination with negative consequences suggests that pigs never be purposely exposed to multiple strains of similar virulence.

References - refereed

1. Kapur V, Elam MR, Pawlovich TM, Murtaugh MP. Genetic variation in porcine reproductive and respiratory syndrome virus isolates in the Midwestern United States. J Gen Virol. 1996;77:1271-1276.

3. Mengeling WL, Lager KM, Vorwald AC. Clinical consequences of exposing gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of "atypical" PRRS. Am J Vet Res. 1998;59:1540-1544.

4. Wesley R, Mengeling WL, Lager KM, Vorwald AC, Roof MB. Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus. Am J Vet Res. 1999;60:463-467.

6. Yuan S, Nelsen CJ, Murtaugh MP, Schmitt BJ, Faaberg KS. Recombination between North American strains of porcine reproductive and respiratory syndrome virus. Virus Res. 1999;61:87-98.

7. Joke JFA, van Vugt JJ, Storgaard T, Oleksiewicz MB, Botner A. High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity. J Gen Virol. 2001;82:2615-2620.

10. Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol. 2000;74:309-329.

11. Mengeling WL, Lager KM, Wesley RD, Clouser DF, Vorwald AC, Roof MB. Diagnostic implications of concurrent inoculation with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus. Am J Vet Res. 1999;60:110-122.

12. Mengeling WL, Vorwald AC, Lager KM, Clouser DF, Wesley RD. Identification and clinical assessment of suspected vaccine-related field strains of porcine reproductive and respiratory syndrome virus. Am J Vet Res. 1999;60:334-340.

13. Brown SM, Subak-Sharpe JH, Harland J, MacLean AR. Analysis of intrastrain recombination in herpes simplex virus type 1 strain 17 and herpes simplex type 2 strain HG52 using restriction endonuclease sites as unselected markers and temperature-sensitive lesions as selected markers. J Gen Virol. 1992;73:293-301.

14. Rowland RR, Steffen M, Ackerman T, Benfield DA. The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332. Virology. 1999;259:262-266.

References - non refereed

2. Zimmerman JJ, Epperson W, Wills RW, McKean JD. Results of the recent survey of the membership of the AASP for outbreaks of sow abortion and mortality. Swine Health Prod. 1997;5:74-75.

5. Mengeling WL, Lager KM, Vorwald AC, Wesley RD, Clouser DF. An update of research at the National Animal Disease Center on current field strains of porcine reproductive and respiratory syndrome (PRRS) virus. Proc Allen D Leman Conf. 1997;24:138-145.

8. Mengeling WL, Clouser DF, Vorwald AC, Lager KM. Genetic recombination among strains of porcine reproductive and respiratory syndrome virus. Proc Conf Res Workers in Anim Dis. 2000. Abstract 134.

9. Mengeling WL, Lager KM, Vorwald AC. An overview on vaccination for porcine reproductive and respiratory syndrome. Proc Allen D Leman Conf. 1996;23:139-146.