High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity
Joke J. F. A. van Vugta,1,
Torben Storgaardb,1,
Martin B. Oleksiewicz1 and
Anette Bøtner1
Danish Veterinary Institute for Virus Research, Lindholm, DK-4771 Kalvehave, , Denmark1
Author for correspondence: Torben Storgaard. Fax +45 44 43 45 58. e-mail TStS{at}novonordisk.com
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Abstract
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Two types of porcine reproductive and respiratory syndrome virus (PRRSV) exist, a North American type and a European type. The co-existence of both types in some countries, such as Denmark, Slovakia and Canada, creates a risk of inter-type recombination. To evaluate this risk, cell cultures were co-infected with either a North American and a European type of PRRSV or two diverse types of European isolate. Subsequently, an approximately 600 bp region of the PRRSV genome was tested for recombination by quantitative real-time RTPCR. Between 0·1 and 2·5% RNA recombination was found between the European isolates, but no recombination was detected between the European and North American types. Calculation of the maximum theoretical risk of EuropeanAmerican recombination, based on the sensitivity of the RTPCR system, revealed that RNA recombination between the European and North American types of PRRSV is at least 10000 times less likely to occur than RNA recombination between diverse European isolates.
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Introduction
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After the initial peak of porcine reproductive and respiratory syndrome (PRRS) outbreaks in 19871991 (Keffaber, 1989
; Wensvoort et al., 1991
), the disease is now endemic in North America, Europe and Asia. Until recently, the European type of PRRS virus (PRRSV) was restricted to Europe, while the American type of PRRSV was restricted to North America and Asia. Now, however, European wild-type PRRSV has been isolated from pigs in North America (Dewey et al., 2000
) and American wild-type PRRSV has been isolated in Slovakia (Psikal et al., 1999
). Furthermore, in several European countries, a modified, live vaccine based on the American type of PRRSV has been used and, at least in Denmark, this live vaccine has reverted to wild-type virus (Bøtner et al., 1997
; Nielsen et al., 2001
; Storgaard et al., 1999
). Recombination between the American isolates of PRRSV (intra-type recombination) occurs (Yuan et al., 1999
), so the co-existence of the European and the American type of PRRSV within herds and even within single animals, as seen in some countries such as Denmark (Anette Bøtner, personal communication), creates a potential risk for inter-type recombination. This could result in serious problems with virus typing and vaccination and might potentially even generate a virus with new biological properties.
To examine inter- and intra-type PRRSV RNA recombination, MARC-145 cells were co-infected (m.o.i. of 0·01 TCID50 per cell) with the Danish 111/92 isolate (Madsen et al., 1998
) and the American PRRS live vaccine virus (Ingelvac PRRS Vet) (Boehringer Ingelheim) or co-infected with the Danish and Dutch Lelystad isolates of PRRSV (Meulenberg et al., 1993
). The Lelystad and Danish isolates were adapted to MARC-145 cells by serial passage. For control infections, MARC-145 cells were mock-infected with medium or infected solely with the Danish, Lelystad or American isolate. After 3 days of incubation at 37 °C, cells were lysed and total RNA was isolated. cDNA was synthesized using PRRSV-specific primers, essentially as described previously (Oleksiewicz et al., 1998
). Detection of viral RNA recombination by PCR requires primers that are absolutely specific for each of the three virus isolates. Danish-specific forward (5' GCGTCACTTTCAACAAGCCATCTC) and reverse (5' TTTGATGGTAACAAGGTCGCTGC) primers that amplified an expected fragment of 894 bp only when used on cDNA from Danish PRRSV-infected cells were designed (Fig. 1
, lanes 25). Lelystad-specific forward (5' GGTCTCAGCAGCGCAAGAGAA) and reverse (5' CAAATCCTGCAGTGGATACAGCG) primers that amplified an expected fragment of 687 bp only when used on cDNA from Lelystad PRRSV-infected cells were also designed (Fig. 1
, lanes 68). Finally, American-specific forward (5' GCGATAGGGACACCTGTGTATGTT) and reverse (5' GGTAGACACAGTGACTAAAGCGACT) primers that amplified an expected fragment of 626 bp only when used on cDNA from American PRRSV-infected cells were designed (Fig. 1
, lanes 911). All three forward primers were located within the 3' end of open reading frame (ORF) 3 and all three reverse primers were located within the 3' end of ORF 5 (Fig. 2
). PCR was carried out using AmpliTaq Gold polymerase according to the manufacturers instructions (Applied Biosystems), starting with a hot-start activation step of 5 min at 95 °C, followed by 45 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 15 s. To exclude the possibility that artificial recombination might have occurred during RNA isolation, cDNA synthesis or PCR amplification, DanishAmerican and DanishLelystad mixture controls were produced by combining cell lysates from the single PRRSV control infections in a 1:1 ratio before RNA isolation. To detect European intra-type recombination, PCR was carried out using the Danish-specific forward primer and the Lelystad-specific reverse primer on cDNA from DanishLelystad co-infected cells and from the DanishLelystad mixture control. A band with the expected size of 667 bp was seen only on cDNA from co-infected cells and not from the corresponding mixture control (Fig. 1
, lanes 1214). When the equivalent experiment was performed to detect EuropeanAmerican inter-type recombination, no specific band was detected in any of the reactions (Fig. 1
, lanes 1517). It was concluded, therefore, that intra- but not inter-type recombination occurred under the in vitro cell culture conditions used.

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Fig. 1. PCR using homologous or heterologous primer pairs. Lanes 1 and 18 are marker lanes containing X174 HaeIII-digested DNA. The size (bp) of the marker bands is indicated on the left. The primers used for PCR were the Danish (lanes 25), Lelystad (Dutch) (lanes 68) or North American (lanes 911) forward and reverse primer pairs. The Danish forward and Lelystad reverse primers (lanes 1214) and the Danish forward and North American reverse primers (lanes 1517) were also used. The results of PCR on cDNA from cells infected with the Danish (lanes 2, 7 and 10), Lelystad (lanes 3 and 6) and American (lanes 4 and 9) isolates are shown. The results of PCR on cDNA from cells co-infected with the Danish and Lelystad isolates (lane 12), a lysate mixture from cells singly infected with the Danish and Lelystad isolates (lane 13), cells co-infected with the Danish and American isolates (lane 15) or a lysate mixture from cells singly infected with the Danish and American isolates (lane 16) are shown. Lanes 5, 8, 11, 14 and 17 show the result of PCR on cDNA from mock-infected cells. The PCR products are 894 bp (lane 2), 687 bp (lane 6), 626 bp (lane 9) and 667 bp (lane 12) in size. DK, Danish; NL, Lelystad; US, North American; Neg., negative control; Mix cont., mixture control.
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To determine sites of recombination, the DanishLelystad recombinant PCR product was gel-purified and cloned into the pCRII vector (Invitrogen), according to the manufacturers instructions. Plasmid DNA purified from 13 individual clones was sequenced using BigDye chain terminators, as recommended by the manufacturer (Applied Biosystems), and the result was compared to both the sequence of the Danish 111/92 isolate (GenBank accession no. AY034879) and the sequence of the Lelystad isolate (GenBank accession no. M96262). All 13 sequences obtained have been submitted to GenBank (accession nos AF315699AF315711). Seven of the clones had recombined in a 104 bp region in ORF 5, with complete identity between the parental strains (Fig. 2
). Two clones had recombined in a region of 52 bp in ORF 4, with complete identity between the parental strains. Three clones had recombined in 5 (ORF 4), 8 (ORF 4) and 26 (ORF 5) bp regions, with complete sequence identity between the parental strains. Finally, one clone differed from the rest in that two deletions had been introduced at the site of recombination (Fig. 2
), suggesting that the mechanism of recombination is not always precise, as described previously for other RNA viruses (Nagy & Bujarski, 1995
). The Danish and Lelystad PRRSV isolates have 94% overall identity in the region examined for recombination (621 bp), while the Danish and American PRRSV isolates only have 60% overall identity in the region examined for recombination (579 bp). Despite the low level of overall identity between the Danish and American isolates in the 579 bp area, small regions (up to 11 bp) of complete identity do exist. The presence of recombination between the two European isolates in short stretches of identity (less than 11 bp) and the absence of recombination between the Danish and American isolates suggest that short stretches of perfect identity should be flanked by longer regions with a high percentage of identity in order to recombine (Fig. 2
). The total absence of RNA recombination between the Danish and American PRRSV isolates could, however, be caused by insufficient sensitivity of our RTPCR assay or by non-optimal experimental conditions for virus recombination. To evaluate these two possibilities, a quantitative real-time RTPCR was established for the different viral RNA targets. This was carried out by adding SYBR Green I (375000x concentration, diluted in the final PCR) (Molecular Probes) to the PCR set-up. SYBR Green I increases its fluorescence upon binding to the PCR product (double-stranded DNA) and this increase in fluorescence was measured in each PCR elongation step using an ABI 7700 thermal cycler (Applied Biosystems). For this real-time PCR, the specificity of the primers was further increased by the addition of Perfect Match PCR Enhancer (Stratagene) to a final concentration of 1 mU/µl. To determine the quantitative nature of the five different PCR tests (Danish, Lelystad, American, DanishLelystad and DanishAmerican), PCR products were produced for each of the target sequences using their respective primer set and cloned into the pCRII vector. As no DanishAmerican PCR product was produced under the experimental conditions used, this PCR product was made artificially using internal primers with overlap extension, as described by others (Horton et al., 1989
). Tenfold dilutions were then produced for each of the five plasmid controls, spanning the range of 1108 DNA copies per reaction. Real-time PCR was performed in triplicate and the logarithm of the number of DNA copies per sample was plotted as a function of the PCR cycle at which the fluorescence increased above the background level (CT value) (Ririe et al., 1997
). This resulted in a nearly perfect linear correlation (r2>0·99) for all five different primer combinations, showing that all primer combinations resulted in a quantitative PCR with a linear range of eight orders of magnitude (data not shown). Moreover, the CT value versus the copy number plots allowed us to compensate for the slightly different amplification efficiencies of these five PCR tests (data not show). We then used the quantitative PCR set-up to calculate the percentage of recombination in co-infected cell cultures. The percentage of recombination was calculated by dividing the number of recombinant molecules by the average number of non-recombinant molecules in the sample. To find the optimal conditions for RNA recombination, cell cultures were infected with different virus ratios and harvested at different time-points. In a total of 43 experiments, the percentage of European intra-type RNA recombination in the cell lysate ranged from 0·1 to 2·5%, with no apparent correlation to the amount or ratio of virus used. Also, the cell supernatant was investigated for the presence of intra-type recombinant RNA. At 6 and 24 h after infection, no RNA recombination between the two European isolates could be detected in the supernatant. At 48 and 72 h after infection, between 0·1 and 0·9% RNA recombination was detected in the supernatant from cells co-infected with the Danish and Lelystad isolates (Fig. 3A
). This percentage of European intra-type recombination found in the current study might be slightly less than the percentage of recombination reported previously for other arteriviruses. Among two North American PRRSV isolates, up to 10% RNA recombination was found in a 1182 bp region (Yuan et al., 1999
) and for lactate dehydrogenase-elevating virus, up to 5% RNA recombination was found in a 1276 bp region (Li et al., 1999
). The current study is the first to show recombination in the European type and also, it is the only one that has quantified precisely the level of RNA recombination. The fragment investigated for recombination in the current study is almost half the size of those reported previously for arterivirus recombination (Li et al., 1999
; Yuan et al., 1999
) and it would be expected that the relative amount of recombination is proportional to the length of the fragment studied. It is interesting to speculate that if there is up to 2·5% recombination in a 621 bp fragment, the level of RNA recombination for the complete genome of 15000 bp would be up to 60%, assuming an evenly distributed level of sequence identity across the whole viral genome. That does not even take into account double crossovers undetected by the current assay (Baric et al., 1990
) and also does not account for the fact that the present study only measured DanishLelystad recombination and not LelystadDanish recombination. Of course, it is highly speculative, but, nevertheless, the calculations suggest that, on average, more than one recombination event might take place per viral genome per cell culture passage.
All tested virus ratios between the Danish and Lelystad isolate resulted in RNA recombination, showing that the current assay was robust at generating recombinant RNA. The same quantitative RTPCR methodology was therefore applied to study whether there was recombination between the European and North American types of PRRSV. Under none of the conditions tested was recombination observed (Fig. 3B
). Nevertheless, the quantitative real-time PCR allowed us to determine the sensitivity of the assay to detect inter-type recombination by comparing the minimal amount of recombinant RNA that could have been detected with the amount of the parental virus RNA present in the co-infected cells. It was calculated that if inter-type recombination was present at a low level, below the sensitivity of the RTPCR, recombination occurred at a frequency of less than 10-6. The maximum theoretical risk of inter-type recombination seems therefore to be at least 10000 times less likely to occur than European intra-type recombination.
Because recombination destroys molecular clocks (Schierup & Hein, 2000
), the recent demonstration of a perfect molecular clock in the European type of PRRSV (Forsberg et al., 2001
; Oleksiewicz et al., 2000
) indicates that recombination among Danish isolates of PRRSV has been a rare event in the field. Taken together with the result of the current study, the risk of inter-type recombination in the field might seem very low. Furthermore, even if inter-type RNA recombinants are generated at a low level under field conditions, there is a good chance that such recombinants are not viable due to the low level of similarity between the parental strains (Godeny et al., 1993
) or that inter-type recombination would be out competed by one of the original virus isolates, as seen for the recombinants of the American PRRSV isolates in cell culture (Yuan et al., 1999
). However, if an inter-type recombinant virus had a selective advantage due to, for example, pre-existing immunity to the parental viruses in the pig population, even an extremely rare non-homologous recombination event could rapidly spread in the pig population. It is important, therefore, that the risk of new inter-type PRRSV recombinants is kept in mind constantly when evaluating current and new diagnostic procedures.
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Acknowledgments
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Preben Normann is thanked for the introduction to the automatic DNA sequencer. This project was partially supported by a grant (BIOT99-2) from the Danish Ministry of Food, Agriculture and Fishery given to Torben Storgaard. Joke van Vugt did the experimental work as part of her Masters degree in biology at the Wageningen University in The Netherlands.
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Footnotes
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The GenBank accession nos of the sequences reported in this paper are AF315699AF315711 and AY034879.
a Present address: Laboratory of Entomology, Bode 49, Postbus 9101, 6700 HB Wageningen, The Netherlands. 
b Present address: Novo Nordisk A/S, Novo Nordisk Park, DK-2760, M
løv, Denmark. 
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