Phenotypic characteristic of wild-type and vaccine viruses in MARC-145.
The vaccine strains (MLV and ATP) grew more efficiently than the wild-type parental viruses (VR2332 and JA142) in MARC-145 cells (Fig. ). The progeny virus titers of all four viruses were similar up to 12 h after inoculation. After 12 h p.i., MLV and ATP started to replicate much faster than their wild-type parental strains as the vaccine viruses produced a higher titer of progeny viruses. At 48 h p.i., the progeny virus titer from cells inoculated with the vaccine viruses reached 106.5 TCID50/0.1 ml, whereas the progeny virus titer from cells inoculated with the wild-type parental strains was approximately 104.5 TCID50/0.1 ml (P < 0.001).
FIG. 1. Different growth characteristics of wild-type PRRSVs (VR2332, JA142) and their cell-attenuated vaccine viruses (MLV, ATP) in MARC-145 cells as measured by multistep growth (A) and one-step (B) curves and immunofluorescence microscopy (C). Sup, supernatant. (more ...)
Similar results were observed in the one-step growth curve and immunofluorescence assessment of virus replication. The infectious progeny virus titers of VR2332 and MLV in both the culture fluid fraction (i.e., cell-free form) and the cell fraction (cell-associated form) were similar for each virus during the observation period. However, the kinetics of progeny virus production were significantly different between the two viruses. The MLV virus grew rapidly and steadily to 105 TCID50/0.1 ml during 24 h p.i., forming a linear growth curve overall, whereas the VR2332 virus grew slowly and reached a plateau (5 × 102 TCID50/0.1 ml) after 15 h p.i. (Fig. ). In immunofluorescence microscopy, the sizes and numbers of foci produced by VR2332 and MLV were similar until 15 h, but those by MLV increased drastically by 24 h compared to what was seen for VR2332 (Fig. ).
The different growth characteristics of the vaccine viruses and their wild-type parental strains yielded the production of differently sized plaques in MARC-145 cells (Fig. ). The vaccine strains, i.e., MLV and ATP (Fig. ), produced larger plaques (diameter, >4 mm on average), while some field strains (Fig. ) generated medium-sized plaques (diameter, between 2 and 3 mm on average). In contrast, the parental viruses, i.e., JA142 (Fig. ) and VR2332 (Fig. ), produced much smaller plaques (diameter, <1 ml on average) within the same incubation time (4 days) and under the same propagation conditions.
FIG. 2. Photomicroscopy of representative plaques produced by wild-type and attenuated PRRSVs in MARC-145 cells. The vaccine strains or VLVs produce bigger plaques (≥2 mm in diameter) (MLV [A] and 64955-01 [B]), whereas wild-type viruses including the (more ...) Stability of the cell growth phenotypic characteristic of virus.
When the CC-01 strain was sequentially passed in three independent lines of pigs 13 times (a total of 726 days of in vivo replication), the faster growth and higher level of viremia began to be observed at the second passage of the virus (4
). In contrast to the CC-01 virus that produced large-sized plaques (diameter, >4 mm) in MARC-145, its descendants collected from subsequent pig passages started to produce plaques smaller than those by the CC-01 after two or three pig passages (Fig. ). After the third (Fig. , lines A and B) or fourth (Fig. , line C) passage, all recovered progeny viruses produced plaques with diameters of less than 2 mm. Once the plaque size became small, the viruses recovered from subsequent pig passages produced the same small-sized plaques until the termination of pig-to-pig passages.
FIG. 3. The change in the size of plaques produced by a MLV vaccine-like PRRSVs (CC-01) during sequential pig-to-pig passages which were maintained in three independent lines (A, B, and C). Two plaque-cloned viruses isolated from each serum sample collected 7 (more ...)
During sequential cell culture passages of the VR2332 and JA142 viruses, the size of plaques produced by the viruses began to get bigger after 17 passages. However, overall plaque size still remained smaller than 2 mm in diameter, indicating that the cell growth phenotype (i.e., smaller-sized plaque) of wild-type PRRSVs is relatively stable during cell passages.
Genetic, antigenic, and phenotypic characteristics of PRRSV field isolates.
Eighty-three field isolates were assayed for their susceptibilities to antisera raised against VR2332 or JA142. Then, the genetic relatedness of the field isolates to VR2332 and JA142 was assessed based on the ORF5 sequence. Six genotypic clusters were identified among the isolates examined. Twenty-five viruses (≥96.5% homology to VR2332/MLV) and 10 viruses (≥98.5% homology to JA142/ATP) were classified into clusters related to VR2332/MLV and JA142/ATP, respectively (Fig. ). In the VN test, the infection of MARC-145 cells by 17 of the 25 VR2332/MLV-like virus and 9 of the 10 JA142/ATP-like viruses was significantly affected (P < 0.05) by VR2332 or JA142 antiserum, respectively (i.e., a less than fourfold decrease in the susceptibility of tested virus to antisera compared to that of the control viruses, VR2332 or JA142). In contrast, the infectivities of the remaining 48 viruses to MARC-145 were not significantly affected by the VR2332 or JA142 antiserum.
FIG. 4. Phylogenetic relationship of 83 PRRSV field isolates with the vaccine viruses (MLV, ATP) and their parental viruses (VR2332, JA142) based on ORF5 nucleotide sequence. Boldface and asterisks indicate isolates whose infections were significantly affected (more ...)
Thirteen of the 17 VR2332/MLV-like viruses and all 9 JA142/ATP-like viruses produced medium-sized (2- to 3-mm) to big-sized (>3-mm) plaques, which were similar to those produced by the MLV and ATP strains. Therefore, these 22 viruses were defined as VLVs, since they were antigenically and genetically close to VR2332/MLV or JA142/ATP and still maintained the phenotype of the vaccine virus (i.e., bigger-sized plaques). All 61 field isolates except one produced small-sized plaques (<2 mm), similar to those produced by VR2332 and JA142. Interestingly, the one virus designated 2M11715 was not closely related to either VR2332 or JA142 (90.9% or 90% ORF5 nucleotide homology to each virus) and produced medium-sized plaques (2.5 mm).
Relationship between phenotypic and genetic similarities of field isolates.
As shown in Fig. and , the genetic proximity of the ORF5 amino acid sequence of the viruses to VR2332 or MLV was not always well correlated with phenotypic characteristics. Five of the 12 viruses that were genetically close to MLV (99.5% homology) produced medium-sized (2- to 3-mm) or big-sized (>3-mm) plaques and the other seven viruses that were closely related to VR2332 (95 to 99.5% homology) produced small-sized plaques (<2 mm). However, the remaining 11 VR2332/MLV-like viruses had the same genetic distance from both VR2332 and MLV. Among those, six viruses (98.5 to 99.5% homology to both viruses) produced medium- or big-sized plaques, whereas the remaining five viruses (95% to 99% homology to both viruses) produced small-sized plaques. In addition, two viruses, which were genetically closer to VR2332 (98 and 99% homology, respectively), produced medium- or big-sized plaques.
FIG. 5. The relationship in plaque size and sequence proximity of between 35 field isolates defined as VLVs and the vaccine strains (MLV and ATP) or their wild-type parental strains (VR2332 and JA142). The horizontal solid line at 2 mm of plaque size indicates (more ...)
In contrast, the genetic proximity of the ORF5 amino acid sequence of the viruses to JA142 or ATP appeared to have a better correlation with phenotypic characteristics. Seven viruses closely related to ATP (98.5 to 99.5% homology) produced a medium- or big-sized plaque, and one virus closely related to JA142 (97% homology) produced a small-sized plaque. Nonetheless, two viruses genetically closer to JA142 based on ORF5 amino acid sequence (98% homology) produced a big-sized plaque, indicating that ORF5 sequence homology may not be able to differentiate VLVs from wild-type viruses.
Genetic markers for PRRSVs with vaccine virus-like phenotype.
By comparing sequences of ORF2 to -7 among vaccine viruses, VLVs, and wild-type viruses, four unique amino acids were identified for each of the MLV and ATP vaccine strains (Tables and ). Phenylalanine at position 10 (F10) in ORF2a and tyrosine at 9 (Y9) in ORF2b, which arose from the same nucleic acid change, glycine at 151 (G151) in ORF5, and glutamate at 16 (E16) in ORF6 were found only in the MLV. Glutamate at 85 (E85) and tyrosine at 165 (Y165) in ORF3, serine at 80 (S80) in ORF5, and cysteine at 62 (C62) in ORF6 were found only in the ATP. Among these unique sequences, F10 in ORF2a and E85 and Y165 in ORF3 were the most stable and consistent sequence elements for MLV-like and ATP-like PRRSVs, respectively, since those amino acids were identified only in the vaccine viruses and VLVs that produced medium- to big-sized plaques among the 83 field isolates examined (Table ).
Unique amino acid sequences for MLV-like viruses
Unique amino acid sequences for ATP-like viruses
Stability of unique amino acid sequences of vaccine viruses or VLVs during passages in animals and MARC-145.
Amino acid sequences F10 in ORF2a and Y9 in ORF2b of the CC-01 strain (MLV-like virus) reverted to the sequences of VR2332, i.e., leucine (L) and aspartate (D), respectively, after three passages in the pigs (lines A and C). In line B, however, F10 was still observed in ORF2a without reversion to L even after 13 sequential pig-to-pig passages of the virus, suggesting that F10 in ORF2a may be stable during in vivo passages of the MLV strain. The Y9 in ORF2b was, on the other hand, changed into histamine (H) instead of D after three passages, which remained unchanged until the end of 13 pig passages. The H9 in ORF2b was found in field isolates which produced a medium- to big-sized plaque (Table ) and thus was considered to be a marker amino acid alternative to Y9.
Similarly, the amino acid sequence E16 in ORF6 was substituted with glutamine (Q) after three passages in the pigs. In line C, however, the E16 was substituted with G instead of Q, which remained without any further alteration until the completion of 13 pig-to-pig passages. Therefore, G16 in ORF6 would be an intermediate form between E and Q, similar to H9 in ORF2b, even though such an amino acid was not found in ORF6 of any of the field isolates examined in this study. In contrast, the amino acid sequence G151 in ORF5 quickly reverted to that of the parental virus, i.e., arginine (R), after the first passage in the pigs.
None of the amino acid sequences unique to the vaccine viruses were found in progeny viruses produced during 20 sequential passages of VR2332 or JA142 in MARC-145. All descendant viruses had the same amino acid sequences as VR2332 or JA142 at the determined sites during the passages, suggesting that over 20 passages might be required to have vaccine-specific sequences at the identified positions.
Biological role of the identified genetic markers.
The identified MLV genetic markers were incorporated into the VR2332 infectious clone (Table ). To characterize the mutants, the multistep growth curve (Fig. ) and the size of plaques they produced (Fig. ) were determined with MARC-145 cells. All of the mutants appeared to grow better than VR2332E which was rescued from the original infectious clone. The effect of those identified sequences on the replication of the virus in MARC-145 cells was, however, accumulative. The growth of mutants with single-site changes (i.e., P2L10F, P5R151G, and P6G16E) was not significantly enhanced compared to that of VR2332E (P = 0.07). In contrast, mutants with changes in two sites (i.e., P25, P26, and P56) grew significantly better than did single-site-changed mutants (P = 0.01) and VR2332E (P = 0.002). Likewise, P256, i.e., a mutant with changes in three sites, grew significantly better than the mutants with changes in two sites (P = 0.008), although its growth was still less than that of MLV.
List of mutants and their mutation profiles
FIG. 6. Growth characteristics of a modified live PRRSV vaccine (MLV), the parental strain of the vaccine (VR2332E), and mutants constructed from a VR2332-based infectious cDNA clone with amino acid substitution(s) in one or more structural proteins of VR2332 (more ...)
A similar observation was also made for the effect of the identified sequences on the size of plaques. The size of the plaques produced by the mutants became bigger and bigger when more of the identified genetic markers for MLV were incorporated into the VR2332 infectious clone (Fig. ).