Abstract
Restriction fragment length polymorphism (RFLP) analysis is one of the tools commonly used to study the molecular epidemiology of porcine reproductive and respiratory syndrome viruses (PRRSVs). As PRRSVs are genetically variable, the stability of the RFLP pattern of a PRRSV during in vivo replication was evaluated by carrying out 13 sequential pig-to-pig passages (P1 to P13) of PRRSV ATCC VR-2332 in three independent pig lines for a total of 727 days. During P1 the pigs were inoculated with a homogeneous inoculum (CC-01) prepared through a series of plaque purifications, and during P2 to P13 the pigs were inoculated with a tissue filtrate from the corresponding pig in the previous passage. Fifteen viral plaque clones were directly isolated from CC-01 and the day 7 serum of each pig of each passage, open reading frame 5 of the clones was sequenced, and the clones were compared to CC-01 to assess the mutation rates and RFLP patterns (obtained by digestion with MluI, HincII, and SacII) over time. Among the 495 viral clones recovered during the passages, 398 clones, including CC-01, had pattern 2-5-2 (MluI-HincII-SacII); however, the remaining 97 viral clones showed different patterns (2-6-2 [P2], 1-5-2 [P3], 2-5-4 [P7], and 2-1-2 [P10]). Importantly, the MluI site that was reported to be present in only one of the PRRS modified live virus vaccine strains (Ingelvac) and its parental strain (ATCC VR-2332) can disappear during in vivo replication. Furthermore, sequence homology between CC-01 and clones with pattern 2-5-2 or clones with other patterns differed by 0.05 to 1.58% and 0.5 to 1.45%, respectively, suggesting that RFLP analysis cannot accurately predict genetic relatedness between PRRSVs. Collectively, precaution should be taken when the molecular epidemiology of PRRSVs is evaluated by RFLP analysis.
Porcine reproductive and respiratory syndrome (PRRS) is a plague causing significant economic loss to pig production throughout the world. The PRRS virus, the causative agent, is an RNA virus belonging to the family Arteriviridae (4, 22) and has shown several outstanding characteristics that are strong impediments to effective disease control. Those characteristics are well reviewed in the 2003 PRRS compendium (28). One of those is that PRRS virus continues to change, as evidenced by the high degrees of genetic and antigenic variability that exist among field isolates (2, 8, 10, 11, 17, 18). Such a high degree of heterogeneity among PRRS viruses prompted the development of molecular tools such as restriction fragment length polymorphism (RFLP) analysis (25), sequencing (2, 8, 10, 11, 13, 14, 17, 21), and heteroduplex mobility assay (12) for use in further characterizing the genotypes of PRRS viruses and tracing virus movements within or among herds. All of these methods target open reading frame 5 (ORF5) of PRRS virus, which encodes the major envelope (gp5) protein (15, 16, 19), since ORF5 has shown the highest genetic variability among PRRS viruses compared to the variabilities of other genes (2, 10).
Among the molecular tools available, RFLP analysis is a rapid and relatively inexpensive nucleic acid-based differential test; hence, the technique has been used in numerous field-based molecular epidemiological surveys of PRRS viruses reported previously (3, 6, 9, 24). RFLP analysis of PRRS virus starts with PCR amplification of ORF5, followed by digestion of the PCR product with the three restriction enzymes MluI, HincII, and SacII. Then, a three-digit code, based on the pattern of cutting by each enzyme, in the order MluI, HincII, and SacII, is assigned to the virus tested. If necessary, additional restriction enzymes can be added for further characterization of the virus.
The foundation for the popular use of RFLP analysis is that the RFLP pattern of a PRRS virus is relatively stable during infection, although the potential for change was suggested (25, 26). However, field observations demonstrated the emergence of new RFLP patterns and the disappearance of existing RFLP patterns over a period of time (3). Furthermore, a high level of genetic variability was shown to exist among field isolates of PRRS virus with the same RFLP pattern of cutting (27). These observations raised concern over the usefulness of RFLP analysis for tracking virus movements and predicting genetic relatedness among PRRS viruses. The study described here was conducted to evaluate the stability of the RFLP pattern of a PRRS virus during in vivo replications.
MATERIALS AND METHODS
Virus.
The North American prototype PRRS virus, ATCC VR-2332 (American Type Culture Collection [ATCC], Manassas, Va.) was used in the study. To produce a highly homogeneous challenge virus, ATCC VR-2332 was subjected to three rounds of plaquing on MARC-145 cells. Following the third round of plaquing, one virus clone, designated VR-2332-CC-01 (hereafter referred to as CC-01), was selected and propagated once on MARC-145 cells to obtain an amount sufficient to inoculate the pigs in passage 1 (P1). Surplus CC-01 was divided into aliquots and stored at −80°C for future use.
Fifteen viral clones were obtained by plaque cloning from CC-01, and the ORF5 sequence was determined to assess the variability of the virus population within the inoculum and provide a baseline for comparison. The CC-01 inoculum was determined to be homogeneous, as 15 viral plaque clones isolated from the inoculum shared 100% sequence homology (5).
Experimental design.
The experiment began by inoculating PRRS virus-naïve pigs with highly homologous clone CC-01. Three independent lines of pigs (lines A, B, and C) were established for in vivo virus replication, and the pigs were monitored over 13 animal passages (P1 to P13), representing a total of 727 days of in vivo replication. Pigs in a fourth line (line D) served both as mock-infected negative controls and as environmental sentinels. Pigs 1A, 1B, and 1C were inoculated with CC-01, and during P2 to P13 pigs were inoculated with a tissue filtrate from the corresponding pig in the previous passage. The time between consecutive passages was 60 days. At each passage, serum samples were periodically collected from all pigs and were used to monitor viremia and the antibody response to PRRS virus. In addition, 15 viral plaque clones were directly isolated from the day 7 serum of each pig of each passage and sequenced for genetic characterization of ORF5. The RFLP pattern of each viral clone obtained by digestion with three restriction enzymes (MluI, HincII, and SacII) was initially predicted from sequence data based on the previously published coding system (25). Viral clones with distinct patterns were again examined by RFLP analysis, as originally reported, to confirm the patterns.
Animal and animal care.
Crossbred pigs were obtained from a closed specific-pathogen-free herd known to be free of PRRS virus. Pigs were weaned at 10 to 14 days of age and were housed in groups in HEPA-filtered isolation units for several days prior to exposure to the virus and were then housed individually in the isolation units from the time of exposure until the end of the observation period. Throughout the study, the animals were housed and cared for in compliance with the requirements given in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (7).
Sequencing and RFLP analysis.
Total RNA was extracted from each virus clone by using the QIAamp viral RNA mini kit (Qiagen, Inc., Valencia, Calif.) by following the protocol recommended by the manufacturer. The entire ORF5 of PRRS virus was amplified by reverse transcription-PCR from 10 μl of RNA extracted by using a OneStep reverse transcription-PCR kit (Qiagen) and primers P5F (5′-CCTGAGACCATGAGGTGGG-3′) and P5R (5′-TTTAGGGCATATATCATCACTGG-3′). The primers were developed by using the sequence information for PRRS virus strain ATCC VR-2332 (GenBank accession no. PRU87392) and were designed to amplify the entire ORF5 region as well as the flanking ORF4 and ORF6 regions. Reverse transcription was performed for 30 min at 50°C. Reverse transcriptase was inactivated, and Taq polymerase was activated by raising the temperature to 95°C for 15 min. PCR amplification was done with 35 cycles of denaturation at 94°C for 15 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s. After the last cycle, the extension period was maintained at 72°C for another 2 min. PCR products (764 bp) were purified with either a QIAquick PCR purification kit or a QIAquick 96 PCR purification kit (Qiagen) by following the procedures recommended by the manufacturer. The final purified products (30 μg/ml each) and primers P5F and P5R (5 pmol each) were submitted to the Iowa State University Nucleic Acid Facility, where sequencing was performed.
For RFLP analysis, each purified product was incubated at 37°C for 2 h with MluI, HincII, or SacII (New England Biolabs, Inc., Beverly, Mass.). The digested materials were then electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. The RFLP pattern of each viral clone was determined on the basis of the molecular masses of the resulting products according to the coding system proposed by Wesley et al. (25).
Analysis of data.
The sequences of the viral clones were analyzed with DNASTAR software (DNASTAR Inc., Madison, Wis.) and were compared to the sequence of CC-01 to evaluate the degree of mutation over time. Sequence alignment was done with the Clustal W program (23). The nucleotide sequences of ATCC VR-2332 (20) and the Ingelvac PRRS (Boehringer-Ingelheim Vetmedica, Inc., St. Joseph, Mo.) vaccine strain (1) were obtained from GenBank and included in the analyses. The mutation rates were calculated as the proportion of substitutions and were expressed as the mean percent change in the sequence compared with the sequence of plaque-cloned viruses.
RESULTS
PRRS viruses in line A could not be passed to subsequent pigs after P7, while the viruses in lines B and C were successfully passed to subsequent pigs until the end of the study (i.e., P13). A total of 495 viral clones (15 clones per pig line at each passage) were recovered from a total of 33 pigs (i.e., line A, n = 7 pigs; line B, n = 13 pigs; and line C, n = 13 pigs) over 13 passages (i.e., 727 days of in vivo replication) and sequenced. In agreement with a previous report (5), the ORF5 nucleotide sequence of CC-01 continued to change over time, and different lines (i.e., lines A, B, and C) showed different mutation rates, as illustrated in Fig. 1. The ORF5 sequence of the inoculum virus, CC-01, annually deviated, on average, 0.75, 0.79, and 0.73% in lines A, B, and C, respectively, under the conditions present in this study. While some nucleotide substitutions (at positions 32, 56, 100, 101, 207, 281, 390, 399, 451, 453, and 513) remained throughout the study, once they occurred, most other changes randomly appeared, disappeared, and reappeared during the study. Over time the mutation rates (Y values) of ORF5 of CC-01 in lines A, B, and C were YA = 0.112x + 0.082 (r2 = 0.807), YB = 0.104x + 0.114 (r2 = 0.89), and YC = 0.114x + 0.083 (r2 = 0.772), respectively, where x represents the number of passages. It is interesting that the regression coefficient (r2) value for line C was lower than those for lines A and B because line C showed a “yo-yo” phenomenon in the mutation rate; i.e., between passages there were increases and decreases in mutations instead of a consistent increase.
FIG. 1.
Change of nucleotide sequence in ORF5 during sequential passages in pigs of a plaque-cloned PRRS virus (CC-01) derived from ATCC VR-2332. Each symbol represents the mean mutation rate for 15 plaque-cloned isolates obtained from each pig serum sample collected at day 7 postinoculation for each passage. The mutation rate at each time was calculated as the proportion of nucleotide substitutions in the ORF5 sequence of each cloned virus in comparison to the sequence of the inoculum (CC-01). Error bars are the standard errors of the mean.
During the study, a total of 86 nucleotide variants (NVs) were derived from CC-01, and these corresponded to 39 amino acid variants. The phylogenetic relationship among the 86 NVs is illustrated in Fig. 2, demonstrating that the phylogenetic distance of each NV from CC-01 is correlated with the chronological order of their appearance during the study. That is, NVs appearing in an earlier passage showed a shorter phylogenetic distance (i.e., fewer genetic changes) from the inoculum virus. The CC-01 genotype completely disappeared after P1. At each passage, a particular NV became the dominant genotype among plaque-cloned virus isolates. Interestingly, most of the NVs observed went through a negative selection (i.e., disappeared) in the subsequent passage. However, a few NVs (e.g., NV-01, NV-23, NV-34, NV-37, NV-40, and NV-63) remained for several subsequent passages once they initially appeared.
FIG. 2.
Phylogenetic relationship of plaque-cloned PRRS virus isolates representing different NVs and RFLP patterns that appeared during 727 days of replication in pigs based on the ORF5 nucleotide sequence. CC-01 is the inoculum virus. Different nucleotide variants are designated with different numbers. The numbers in parentheses indicate the passage number during which the given NV first appeared. NVs without a specific RFLP pattern have pattern 2-5-2.
The predicted RFLP patterns of the viral clones are summarized in Table 1. Among the clones, 398 clones (80.4%) were predicted to have pattern 2-5-2, which is the same as the RFLP pattern of the inoculum virus (CC-01). The remaining 97 viral clones were predicted to have RFLP patterns different from that of the initial inoculum. The appearance of different RFLP patterns varied between passages and among pig lines, suggesting that mutational changes in PRRS viral ORF5 are likely random and independent. Despite the incidence of randomness and independence, the complete conversion and reversion of the RFLP patterns among all 15 plaque-cloned viruses were observed between P11 and P12 and between P7 and P8 in lines B and C, respectively. The biological significance of this phenomenon was not, however, apparent with respect to the clinical responses, including viremia and antibody production (data not shown).
TABLE 1.
RFLPs of ORF5 of PRRS virus that was plaque cloned from ATCC VR-2332, the North American prototype, during a long-term in vivo replication
| Passage | Line | No. of cloned viruses | No. of clones with the following RFLP patterna:
|
||||
|---|---|---|---|---|---|---|---|
| 2-5-2 | 2-6-2 | 1-5-2 | 2-5-4 | 2-1-2 | |||
| 1 | A | 15 | 15a | ||||
| B | 15 | 15 | |||||
| C | 15 | 15 | |||||
| 2 | A | 15 | 15 | ||||
| B | 15 | 9 | 6 | ||||
| C | 15 | 15 | |||||
| 3 | A | 15 | 15 | ||||
| B | 15 | 14 | 1 | ||||
| C | 15 | 15 | |||||
| 4 | A | 15 | 15 | ||||
| B | 15 | 15 | |||||
| C | 15 | 15 | |||||
| 5 | A | 15 | 15 | ||||
| B | 15 | 15 | |||||
| C | 15 | 15 | |||||
| 6 | A | 15 | 15 | ||||
| B | 15 | 15 | |||||
| C | 15 | 15 | |||||
| 7 | A | 15 | 15 | ||||
| B | 15 | 15 | |||||
| C | 15 | 15 | |||||
| 8 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| 9 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| 10 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| 11 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| 12 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| 13 | B | 15 | 15 | ||||
| C | 15 | 15 | |||||
| Total | 495 | 398 | 6 | 1 | 15 | 75 | |
The RFLP pattern of each virus clone was predicted from its nucleotide sequence.
During the study period (727 days), four RFLP patterns distinct from 2-5-2 were observed: 2-6-2 (P2), 1-5-2 (P3), 2-5-4 (P7), and 2-1-2 (P10). Such predicted patterns were confirmed to be the same as those obtained by the actual RFLP analysis (Fig. 3), demonstrating that the RFLP pattern of each virus predicted from its nucleotide sequence was accurate. While most of the RFLP patterns different from pattern 2-5-2 were short-lived (i.e., they appeared in only one passage and disappeared in the subsequent passage), pattern 2-1-2 became predominant (75 of 97 clones) after nine passages of CC-01. In particular, the RFLP patterns of all viral clones from line C became 2-1-2 (Table 1).
FIG. 3.
A 1.5% agarose gel with ORF5 cDNA of selected plaque-cloned PRRS virus isolates with representative predicted RFLP patterns observed during the 727 days of replication in pigs after digestion with selected restriction enzymes (lanes 1, MluI; lanes 2, HincII; lanes 3, SacII). The PCR product of each virus was digested, electrophoresed along with a DNA ladder (lanes M), and stained with ethidium bromide.
Although the sequences of all three restriction enzyme sites used to characterize the PRRS virus ORF5 changed during the study period, the HincII sites appeared to be more vulnerable to mutational changes than the other two restriction enzyme sites. More importantly, a clone without the MluI site (position 196) that was reported to be present only in Ingelvac PRRS vaccine virus and its parental strain (ATCC VR-2332) but not in wild types (25) appeared during the long-term in vivo replication of CC-01, which was derived from ATCC VR-2332.
Phylogenetically, viral clones with different RFLP patterns, particularly clones with pattern 2-1-2, formed their own cluster which was separated from clusters among viral clones with RFLP pattern 2-5-2 (Fig. 2). However, the overall sequence homology did not provide a clear-cut demarcation between these two groups. The nucleotide sequence divergence between variant viral clones with RFLP pattern 2-5-2 and CC-01 ranged from 0.05 to 1.58%, whereas the sequence discrepancy between viral clones with RFLP patterns other than 2-5-2 and CC-01 ranged from 0.5 to 1.45%. Furthermore, it was observed that virus clones with pattern 2-5-2 that had completely disappeared in the preceding passage reemerged in the successive passage (Table 1); however, their ORF5 genetic makeup was different from that of virus clones with pattern 2-5-2 in an earlier passage.
DISCUSSION
The study described here was conducted to evaluate whether the ORF5 RFLP pattern of a PRRS virus is stable during replication in pigs and how well RFLP analysis of ORF5 can predict the genetic relatedness among PRRS viruses. These questions were important, since RFLP analysis has commonly been used to study the molecular epidemiology of PRRS viruses in the field not only because of its rapid turnaround and inexpensive nature but also because of an assumption that the RFLP pattern of a PRRS virus is stable (25, 26). In contrast to that assumption, the results of this study demonstrated that the ORF5 RFLP pattern of PRRS virus can be changed during in vivo replication and that the change is somewhat unpredictable and inconsistent due to conversion and reversion. The study also demonstrated that the predictive value of a RFLP pattern for genetic similarity or relatedness between PRRS viruses is questionable because of observed continuous genetic changes in ORF5 during the study period (Fig. 1). Although this may not be an unexpected result, since PRRS virus is an RNA virus for which genetic instability is common, to our knowledge this is the first report to prove the instability of the ORF5 RFLP pattern of a PRRS virus in pigs under experimental conditions, thus challenging the reliability of RFLP analysis for rapid genetic characterization.
Several RFLP patterns that differed from that of the inoculum emerged during the 727-day study period (Table 1). All of those patterns have been seen among field isolates implicated in clinical outbreaks of PRRS after the vaccine (Ingelvac PRRS) was introduced to the market (K.-J. Yoon, unpublished data), and some of those were seen as intermediate patterns between the vaccine strain and the wild types even before the vaccine was available (26). As genetic changes in ORF5 were mostly random, no directional change in the RFLP pattern was observed between passages and among pig lines. Furthermore, alteration of the RFLP pattern was not permanent and many times disappeared in a successive passage, although one particular RFLP pattern (pattern 2-1-2) became dominant after nine passages of clone CC-01 (pattern 2-5-2) in pigs. This observation, along with the fact that pattern 2-5-2 was still dominant overall, suggests that there is a purifying process which prevents a radial expansion of mutants until a certain variant(s) among quasispecies acquires a better fitness for the host, although the better adaptation of certain genotypes to the cell culture system used in this study certainly could account for it. Furthermore, complete conversion and reversion of an RFLP pattern between passages could have been attributed to the number (i.e., n = 15) of plaque-cloned isolates from each pig. Although that number was statistically determined at the 95% confidence level for the detection of one variant in a population with 10% deviation, it would be possible that minor variants may be detected if the number of plaque-cloned viruses were increased.
The disappearance of the MluI site from ORF5 of CC-01, which was derived from ATCC VR-2332, during in vivo replication was an unexpected observation. More surprisingly, the virus clone without the MluI site (i.e., pattern 1-5-2) emerged at the early stage in sequential passages, while such a virus clone shared 99.7% sequence homology with CC-01. Previous reports (25, 26) strongly suggested that not all wild-type PRRS viruses have an MluI site in ORF5, unlike the Ingelvac PRRS vaccine virus and parental strain ATCC VR-2332. Consequently, if PRRS viruses were to be evaluated only by RFLP analysis, some viruses without an MluI site could erroneously be categorized into the wrong group, i.e., as the wild type instead of the vaccine type. In fact, the loss of the MluI site appears to occur during natural infection, as field isolates of PRRS virus which have pattern 1-5-2 and which share a relatively high degree of sequence homology (97.0 to 99.3%) with ATCC VR-2332 or the Ingelvac PRRS vaccine strain have been identified (Yoon, unpublished).
In summary, our observations demonstrate that the RFLP pattern of a PRRS virus can be changed during its replication in pigs and that RFLP analysis may not accurately predict genetic similarity or relatedness between or among PRRS viruses. These facts should be taken into consideration when molecular characterization or the epidemiology of PRRS viruses is determined only by RFLP analysis.
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