J Vet Diagn Invest 10:140144 (1998)

Differentiation of a porcine reproductive and respiratory

syndrome virus vaccine strain from North American field
strains by restriction fragment length polymorphism
analysis of ORF 5
Ronald D. Wesley, William L. Mengeling, Kelly M. Lager, Deborah F. Clouser, John G. Landgraf,
Merwin L. Frey
Abstract. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiating
a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain from other North American field
strains was investigated. Open reading frame 5 nucleotide sequence data of the vaccine virus, its parent strain
VR-2332, and 22 other strains of PRRSV included in this study indicated that 3 restriction enzyme gel patterns
characterize the vaccine virus and the parent strain genotype. The combined 3 RFLP patterns differentiate the
vaccine and parent virus from other PRRSV strains. This test will be a valuable tool in epidemiologic studies
and will be useful in identifying individual strains in cases of multistrain PRRSV infections.

Porcine reproductive and respiratory syndrome

(PRRS) is one of the most costly diseases currently
faced by the swine industry. It is characterized clinically by reproductive failure in gilts and sows, weak
and sickly neonatal piglets, and a respiratory disease
of nursery-age and finishing pigs.2 Moreover, many
practitioners believe that infection with PRRS virus
(PRRSV) might potentiate the effects of other respiratory tract bacterial and viral pathogens. Some experimental studies support this belief whereas others
do not.5,6,8,13,14
The viral etiology of PRRS was first determined in
The Netherlands with the isolation of the prototype
Lelystad strain15 and then in the United States with the
prototype American strain, VR-2332.4 Based on genome organization and replication strategy, the
PRRSV is classified as a member of the family Arteriviridae. Structurally, PRRSV is an enveloped virus
that contains a nucleocapsid (N) protein, a nonglycosylated membrane (M) protein, and a surface (E) glycoprotein. The E glycoprotein is encoded by open
reading frame (ORF) 5.11
A commercial modified live virus vaccinea has been
used to vaccinate pigs in the United States since late
1994 and has had some limited use in other countries.
More than 70 million doses have been administered to
pigs by producers and swine practitioners. Although
From the Virology Swine Research Unit, National Animal Disease
Center, USDA, Agricultural Research Service, PO Box 70, Ames,
IA 50010 (Wesley, Mengeling, Lager, Clouser), and the Diagnostic
Virology Laboratory, National Veterinary Services Laboratories,
USDA, Animal and Plant Health Inspection Service, PO Box 844,
Ames, IA 50010 (Landgraf, Frey).
Received for publication August 30, 1997.

the vaccine virus is attenuated, it, like the virulent field

strains of PRRSV, has the ability to persist for at least
several weeks in a vaccinated pig. Consequently the
source of PRRSV isolated from diagnostic samples is
sometimes in doubt.
Here, we describe a differential gel electrophoresis
test that distinguishes a particular vaccine virus from
PRRSV field strains. The test utilizes the reverse transcriptasepolymerase chain reaction (RT-PCR) method
and gives a numerical code to each isolate based on
restriction fragment length polymorphism (RFLP) gel
patterns.
Materials and methods
PRRSV strains were propagated on Marc-145 cells, and
field strains were cloned by 3 rounds of replication at the
end point dilution. The Marc-145 cells9 were cultured in
Earles minimum essential medium, supplemented with 10%
fetal bovine serum and 50 mg/ml gentamicin sulfate in a
humidified 5% CO2 atmosphere at 37 C. The year and location of isolation and the passage history of the original set
of 22 field strains are shown in Table 1. These strains were
isolated during a 6-yr period (19891994) from Canada,
Guatemala, and 13 states in the USA. All of these field
strains were isolated prior to the marketing of the PRRSV
vaccine.a In addition, Table 1 contains the RFLP coding assignments for restriction enzymes MluI, HincII, and SacII.
Viral RNA was isolated for each strain and used as template for RT-PCR. The primers (P420 and P620) and conditions for RT-PCR were as previously described.1 The oligonucleotide primers appear to be universally applicable in
that RNA from all of the North American PRRSVs we have
isolated on MARC-145 cells to date were amplified. The RTPCR gave a DNA fragment of 716 base pairs that contained
PRRSV ORF 5 (603 base pairs in length).
DNA sequencing using Taq polymerase and fluorescently

140
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Differentiation of a PRRS vaccine strain

Table 1.
Virus
designation

1. 46448
2. 46907
3. 1205-D
4. 10654
5. 30093-A
6. 34075
7. 49138
8. 5556
9. 22805
10. 5591
11. 14622
12. 19950-E
13. 26948-2
14. 41572-2
15. 42928
16. 32983-LG
17. 30352-3
18. 47324-2
19. 18310-A
20. 24901
NADC-8 (IA)-92
NADC-9 (IA)-93

141

Origin, passage history, and RFLP code of PRRSV isolates.

Passage history

Origin

Year
isolated

CL-2621

MARC

Total

RFLP
code

IA
KY
MO
IA
IL
NE
TX
MI
KS
NC
AR
MN
VA
NE
IL
NC
MI
Canada
PA
Guatemala
IA
IA

1989
1991
1992
1992
1992
1992
1992
1993
1992
1993
1993
1993
1993
1993
1993
1993
1993
1993
1994
1994
1992
1993

2
unk*
2
2
2
3
unk
unk
2
4
...
...
...
...
...
...
...
...
...
...
...
...

5
5
5
5
5
5
5
5
5
5
7
7
6
7
7
7
10
8
6
7
5
6

7
.5
7
7
7
8
.5
.5
7
9
7
7
6
7
7
7
10
8
6
7
5
6

1-3-2
1-1-2
1-1-2
1-5-1
1-3-2
1-4-2
1-4-2
1-1-4
1-5-3
1-3-2
1-2-4
1-4-2
1-2-4
1-4-4
1-4-2
1-4-2
1-1-4
1-2-4
1-2-2
1-4-2
1-3-4
1-3-4

* unk 5 unknown number of passages.

labeled dideoxynucleotidesb was performed as previously

described.1 ORF 5 sequence data were analyzed by a commercial software packagec to determine restriction enzyme
sites that could distinguish the PRRS vaccine virus from
other PRRSV field strains.

Results
The data indicating that the vaccine virus could be
distinguished were derived from sequence analysis of
ORF 5 of the North American prototype PRRSV
strain, VR-2332, the vaccine strain, and the 22 PRRSV
strains in Table 1, all of which were isolated from the
field prior to the marketing of this vaccine, which was
the first of 2 modified live virus PRRS vaccines.1,12
From these data a set of restriction enzymes, MluI,
HincII, and SacII, was selected for RFLP analysis.
To facilitate the reporting and handling of test data,
each isolate is given a numerical code for its ORF 5
RFLP pattern with enzymes, MluI, HincII, and SacII,
in that order. These RFLP gel patterns are shown in
Fig. 1. MluI either does not cut (code 1) or does cut
(code 2) as is the case for the vaccine virus and
PRRSV VR-2332. HincII has 8 different cutting patterns, including a no cut pattern, which is designated
code 1. SacII has 4 cutting patterns, including a no cut
pattern (code 1). Thus, an example RFLP code for the
vaccine virus is 2-5-2: MluI cuts the PCR product from
the vaccine virus (code 2), the HincII pattern for the
vaccine virus is code 5, and SacII cuts the vaccine
PCR fragment with a code 2 pattern. In this manner,

all PRRSV strains are given a code based on their ORF

5 cutting patterns.
The MluI site in ORF 5 of the vaccine strain and
its parent virus, VR-2332, distinguishes these 2 viruses
from the other PRRSV field strains in Table 1. HincII
differentiated the vaccine and VR-2332 viruses from
20 of the 22 original field strains; only isolates from
Iowa and Kansas (Table 1, no. 4 [IA], no. 9 [KS]) had
the same HincII pattern (code 5) as the vaccine/VR2332 viruses. Although the Iowa and Kansas isolates
had the same HincII RFLP pattern, they could be distinguished from the vaccine/VR-2332 viruses by different SacII RFLP patterns. By analyzing the combined HincII and SacII RFLP patterns, all 22 isolates
in Table 1 could be differentiated a second time from
the vaccine/VR-2332 viruses. Thus, 2 separate RFLP
tests distinguish vaccine/VR-2332 from the isolates in
Table 1. The MluI pattern is 1 RFLP test, and the combined HincII/SacII gel patterns are a second RFLP test.
To further evaluate the RFLP differential test, an
additional 50 prevaccine PRRSV field strains were obtained. These isolates were more regional than the
original 22 strains: 45 were from Iowa, 4 were from
Illinois, and 1 was from Minnesota. All of these viruses were isolated prior to the extensive use of the
PRRS vaccine in late 1994. Forty-two PRRSV isolates
came from the APHIS National Veterinary Services
Laboratory collection, and 8 were isolated at the National Animal Disease Center. All 50 additional strains

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Wesley et al.

Figure 1. General types of RFLP patterns for PRRSV isolates. PCR products (716 bp) containing ORF 5 were digested with restriction
enzymes MluI, HincII, and SacII. There are 2 types of RFLP patterns for MluI, 8 different patterns for HincII, and 4 different patterns for
SacII. A. Restriction enzyme digests were electrophoresed on a 1.8% agarose gel and stained with ethidium bromide. B. A 2% agarose gel
with more band migration was used to resolve HincII patterns 7 and 8. M 5 marker lane with DNA fragments of known size.

were distinguished from the vaccine strain and VR2332 virus by their no-cut MluI RFLP pattern (code
1). Two of the 50 isolates (no. 90 [IA], no. 126 [IA])
had an RFLP code of 1-5-2, indicating that they differed only in their MluI patterns, but each had the same
HincII and SacII patterns as the vaccine strain and VR2332 virus. ORF 5 sequences of these 2 isolates were
aligned and compared using a computer software program.c These 2 isolates were determined to be more
closely related to the vaccine/VR-2332 viruses than to
either no. 4 (IA) or no. 9 (KS), the most closely related
among the original 22 strains (Fig. 3A).
Discussion
Using a series of RFLP patterns, it was possible to
distinguish a PRRSV vaccine strain and the prototype
North American PRRSV, VR-2332, from other
PRRSV field strains. However, a second modified live
PRRS vaccine virusd is not distinguishable. With this
method, PRRSV strains are assigned a 3-digit RFLP
code; the first digit is the MluI RFLP pattern, the
second is the HincII pattern, and the last digit is the
SacII pattern. Thus, the RFLP code for the vaccine

virusa and the VR-2332 virus is 2-5-2, which (based

on sequencing and RFLP data from 90 prevaccine
field strains) is unique to these 2 viruses. These observations are derived from the original 22 PRRSV
strains (Table 1), the 50 additional prevaccine PRRSV
field isolates, and published sequence data from 18
prevaccine PRRSV isolates.1,7,10,12 These data do not
exclude the possibility of other prevaccine wild type
PRRS viruses having 2-5-2 patterns, but probably
they are rare.
Only the vaccine/VR-2332 viruses have an alanine
(A) at residue 137 in a moderately conserved region
of ORF 5.1 The consensus sequence has a serine (S)
at residue 137 (Fig. 2A) for all the PRRSV strains
listed in Table 1 and, in fact, for all PRRSV field
strains examined so far, except for an MO1 isolate.7
This preference for serine at residue 137 explains why
only the vaccine virusa and PRRSV VR-2332 have an
apparently unique MluI site in ORF 5. A G to T transversion at the first base position in codon 137 gives
rise to serine 137 for the original 22 PRRSV strains
(Fig. 2B). There is also a second base change for all
22 strains at the third (wobble) position of codon 137

Figure 2. Residue 137 coding assignment (A) and base substitutions within codons 136 and 137 that alter the MluI site (B). The shaded
bases (A) indicate the 6-nucleotide recognition sequence for MluI, and the arrow (B) shows where MluI cuts.

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Differentiation of a PRRS vaccine strain

143

Figure 3. A. Dendrogram based on ORF 5 nucleotide sequences and the RFLP code for prevaccine PRRSV isolates that cluster and
are known to be more closely related to North American prototype virus VR-2332. ORF 5 nucleotide sequence for RespPRRS, no. 90 (IA)
and no. 126 (IA) were determined for this study and submitted to GenBank with accession numbers AF020048, AF020049, and AF020050.
Other ORF 5 sequences for VR-233212 for isolates no. 4 (IA) and no. 9 (KS),1 for isolates MO1, KS1, MN1, and NE1,7 and for isolates
ISU-22, ISU-179, and ISU-189410 were obtained from GenBank. B. Base substitutions at codon 137 that eliminate the MluI site for these
clustered isolates.

(either G to A or G to T; Fig. 2B). Virus VR-2332 is

an early North American PRRSV; tissue samples containing this virus were collected from a farm in Minnesota in 1989 (J. Collins, personal communication).
Other PRRSV field isolates may have evolved from
VR-2332; thus, serine, rather than the original alanine,
may be the preferred amino acid at residue 137. Another equally likely explanation is that VR-2332 is not
a direct ancestor of current field isolates but represents
an early branch on the main evolutionary tree. In either
case, the reason for a unique MluI site in the vaccine/

VR-2332 viruses is due to the less common presence

of alanine at residue 137.
A dendrogram of PRRSV strains that are most
closely related to the North American prototype virus
VR-2332 is shown in Fig. 3A. All 10 PRRSV strains
in this cluster were isolated before the release of the
PRRS vaccine,a and all lack an MluI site, thus differentiating them from the vaccine strain and VR-2332.
Each of these closely related isolates encodes a serine
at residue 137 because of a G to T transversion at the
first base of codon 137 (Fig. 3B).

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Wesley et al.

The MO1 isolate (from MO, also shown in Fig. 3A)

is more distantly related to this cluster group. The
MO1 isolate was included in this analysis because both
MO1 and VR-2332 contain an alanine at residue 137
(a G at the first base position for the codon); however,
MO1 lacks an MluI site because of 2 other base substitutions at the MluI site. Viruses such as VR-2332
and MO1 with an alanine at residue 137 appear to be
rare, suggesting that serine is the preferred amino acid
in this moderately conserved region of ORF5.1
Although a serine to alanine substitution is a relatively conservative amino acid change, it may be an
important feature for the structure of the glycoprotein
E molecule. Computer-generated secondary structure
profiles3 indicate that glycoprotein E molecules with
an alanine at residue 137 (i.e., VR-2332, RespPRRS,
MO1) have a continuous b sheet through residue 137,
whereas molecules with a serine at residue 137 have
an extra turn in their structure in that region.
At the present time PRRS serology is at a stage
where an individual pig or an entire herd is identified
as having been infected or not. No differential serologic test to distinguish vaccinated animals from fieldvirus-infected pigs exists. Thus, to fill this important
niche, the RFLP test as described here was developed
as a means to differentiate 1 of the 2 modified live
virus vaccine strains from field strains of PRRSV. The
differential test has become widely used because
PRRSV persists in a herd or an individual pig and is
easily isolated. The use of 3 restriction enzymes gives
a high level of assurance of identifying the vaccine
virus even if some genomic change and drift occur in
ORF 5 during virus replication in vivo. Other additional restriction enzymes can also be used to follow
the epidemiology of individual PRRSV strains.
Acknowledgements
We thank Sue Ohlendorf for assistance in preparing this
manuscript. No endorsements are herein implied. Brand
names are necessary to report factually on available data;
however, the USDA neither guarantees nor warrants the
standards of the products, and the use of the names by the
USDA implies no approval of the products to the exclusion
of others that may also be suitable.

Sources and manufacturers

a. RespPRRS/Reproy vaccine, BI/NOBL Laboratories, Sioux Center, IA.
b. Perkin-Elmer, Applied Biosystems Division, Foster City, CA.
c. GeneWorks, version 2.5.1, Intelligenetics, Menlo Park, CA.
d. PrimePac PRRS, Schering-Plough Animal Health, Kenilworth,
NJ.

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