University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Virology Papers
Virology, Nebraska Center for
1-1-2012
Evaluation of monoclonal antibody–based
immunohistochemistry for the detection of
European and North American Porcine reproductive
and respiratory syndrome virus and a comparison
with in situ hybridization and reverse transcription
polymerase chain reaction
Kiwon Han
Seoul National University, Korea
Hwi Won Seo
Seoul National University, Korea
Yeonsu Oh
Seoul National University, Korea
Ikjae Kang
Seoul National University, Korea
Changhoon Park
Seoul National University, Korea
Han, Kiwon; Seo, Hwi Won; Oh, Yeonsu; Kang, Ikjae; Park, Changhoon; Kang, Sang Hoon; Kim, Sung-Hoon; Lee, Bog-Hieu; Kwon,
Byung Joon; and Chae, Chanhee, "Evaluation of monoclonal antibody–based immunohistochemistry for the detection of European
and North American Porcine reproductive and respiratory syndrome virus and a comparison with in situ hybridization and reverse
transcription polymerase chain reaction" (2012). Virology Papers. Paper 227.
http://digitalcommons.unl.edu/virologypub/227
This Article is brought to you for free and open access by the Virology, Nebraska Center for at DigitalCommons@University of Nebraska - Lincoln. It
has been accepted for inclusion in Virology Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.
See next page for additional authors
Follow this and additional works at: http://digitalcommons.unl.edu/virologypub
Part of the Virology Commons
Authors
Kiwon Han, Hwi Won Seo, Yeonsu Oh, Ikjae Kang, Changhoon Park, Sang Hoon Kang, Sung-Hoon Kim,
Bog-Hieu Lee, Byung Joon Kwon, and Chanhee Chae
This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/virologypub/227
Published in Journal of Veterinary Diagnostic Investigation 24:4 (2012), pp. 719–724; doi: 10.1177/1040638712446507
Copyright © 2012 Kiwon Han, Hwi Won Seo, Yeonsu Oh, Ikjae Kang, Changhoon Park, Sang Hoon Kang, Sung-Hoon Kim, BogHieu Lee, Byungjoon Kwon, Chanhee Chae. Published by Sage Publications on behalf of American Association of Veterinary
Laboratory Diagnosticians, Inc. Used by permission.
Evaluation of monoclonal antibody–based immunohistochemistry for
the detection of European and North American Porcine reproductive
and respiratory syndrome virus and a comparison with in situ
hybridization and reverse transcription polymerase chain reaction
Kiwon Han,1 Hwi Won Seo,1 Yeonsu Oh,1 Ikjae Kang,1 Changhoon Park,1 Sang Hoon Kang,2
Sung-Hoon Kim,2 Bog-Hieu Lee,3 Byungjoon Kwon,4 Chanhee Chae 1
1. Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul, Korea
2. College of Oriental Medicine, Kyunghee University, Seoul, Korea
3. Department of Food and Nutrition, Chung- Ang University, Gyeonggi-Do, Korea
4. School of Veterinary Medicine and Biomedical Sciences, Nebraska Center for Virology,
University of Nebraska–Lincoln, Lincoln, NE
Corresponding author — Chanhee Chae, Department of Veterinary Pathology, College of Veterinary Medicine,
Seoul National University, 599 Gwanak-ro, Gwanak-Gu, 151-742, Seoul, Republic of Korea. [email protected]
Abstract.
The objective of the present study was to compare the ability of 2 monoclonal antibodies (mAbs; SDOW17 and SR30)
to detect types 1 and 2 Porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed, paraffin-embedded (FFPE) lung tissues by immunohistochemistry (IHC) and to compare the immunohistochemical results with in
situ hybridization (ISH) and reverse transcription nested polymerase chain reaction (RT-nPCR) detection techniques.
Lungs from 30 experimentally infected pigs (15 pigs with each genotype of PRRSV) and 20 naturally infected pigs
(10 pigs with each genotype of PRRSV) with types 1 and 2 PRRSV, respectively, were used for the IHC, ISH, and RTnPCR analyses. The SR30 mAb-based IHC detected significantly more type 1 PRRSV-positive cells in the accessory
and caudal lobes from the experimentally infected pigs at 7 (P = 0.025) and 14 (P = 0.018) days postinoculation, respectively, compared to the SDOW17 mAb-based IHC. The results demonstrated that SR30 mAb-based IHC is useful for
detecting both types 1 and 2 PRRSV antigen in FFPE lung tissues.
Keywords: Immunohistochemistry, in situ hybridization, pigs, Porcine reproductive and respiratory syndrome virus
Porcine reproductive and respiratory syndrome (PRRS),
caused by the PRRS virus (PRRSV), occurs in 2 clinical
forms, reproductive failure and respiratory disease. Reproductive failure is characterized by abortion and delivery of stillborn near-term fetuses or premature and weak
piglets.27 The respiratory disease is characterized by pronounced hyperpnea, fever, and interstitial pneumonia
in young pigs and mild flu-like signs in growing and finishing pigs.27 PRRSV is a member of the Arterivirus genus (family Arteriviridae, order Nidovirales).4 Genetic analysis has established 2 predominant PRRSV genotypes, type
1 (European genotype) and type 2 (North American genotype), which share approximately 60% nucleotide identity
of the entire genome.1,18
The current diagnostic tests used to determine whether
PRRSV is the cause of respiratory disease are viral isola-
tion, light microscopy, immunohistochemistry (IHC), in
situ hybridization (ISH), reverse transcription polymerase
chain reaction (RT-PCR), and serology.8,15,17 Among these
diagnostic methods, IHC, ISH, and RT-PCR can be applied
to detect PRRSV in formalin-fixed, paraffin-embedded
(FFPE) tissues.8,9
Pathologists prefer to use IHC or ISH to diagnose
PRRSV infection because these 2 methods are useful for the
simultaneous evaluation of histopathological lesions and
the viral loads in lung tissues to determine the degree of severity of respiratory disease. SDOW17 and SR30 monoclonal antibodies (mAbs), which react with the 15-kD nucleocapsid protein of PRRSV by Western blotting analysis,19,25
are the prototypical PRRSV mAbs and are widely and routinely used in IHC to detect PRRSV in FFPE tissues.6-8,12,26
In addition to North America,21 type 1 PRRSV has recently
719
720 Han
et al. in
emerged in Asian countries, including Korea, China, and
Thailand.2,5,16,22 Clinically, a type 2 PRRSV-based vaccine
may not be effective for controlling type 1 PRRSV infection
in pigs23 (Kovacs F, Schagemann G. 2003, “Efficacy of ingelvac® PRRS MLV against European isolates,” in Proceedings
of the 4th International Symposium on Emerging and Re-emerging Pig Diseases, pp. 111–112. Rome). Hence, it is necessary to detect and differentiate between Korean type 1 and
2 PRRSV for proper selection of PRRSV vaccine to control
both genotypes of PRRSV infection effectively in swine
herds. The objective of the present study was 1) to compare
the abilities of 2 mAbs (SDOW17 and SR30) in the detection
of the 2 genotypes of PRRSV in FFPE tissues by IHC and 2)
to compare the immunohistochemical results with ISH and
RT-PCR techniques.
Korean type 1 (SNUVR100057 strain) and type 2
(SNUVR090851 strain) PRRSV used in the current study
were isolated from lung samples from different postweaning pigs in a 1,000-sow herd in Chungcheung Providence
in 2009 and 2010, respectively. This herd has shown severe
respiratory problem in postweaning pigs, aged from 4 to 8
weeks old. The SNUVR100057 strain was identified as type
1 PRRSV based on the nucleotide sequences of open reading frame (ORF)5 (GenBank accession no. JN411262) and
ORF7 (JN411261); the SNUVR090851 strain was identified
as type 2 PRRSV on the basis of the nucleotide sequences of
ORF5 (JN315685) and ORF7 (JN399072).
Thirty-six pigs were purchased from a PRRSV-free herd
and were used at the age of 3 weeks old. All of the pigs
were negative for PRRSV and Porcine circovirus-2 according
to routine serological testing and real-time PCR performed
prior to delivery and again on arrival.10,24 The pigs were
randomly allocated into 2 infected and 1 control groups. In
groups 1 and 2, 15 pigs each were inoculated intranasally
with 3 ml of tissue culture fluid containing 106 50% tissue
culture infective doses (TCID50)/ml of Korean type 1 PRRSV
(second passage in the MARC [cloned African green monkey kidney cell line]-145 cells) or 106 TCID50/ml of Korean
type 2 PRRSV (second passage in the MARC-145 cells). In
group 3, 6 control pigs were exposed in the same manner to
uninfected cell culture supernatants. Ten infected (5 each in
groups 1 and 2) and 2 control pigs from each group were sedated by an intravenous injection of sodium pentobarbital
and then euthanized by electrocution at 7, 14, and 21 days
postinoculation (dpi) as previously described.3 Tissues were
collected from each pig at necropsy. Lungs were collected
for viral isolation from all of the infected and negativecontrol pigs used in the study, as previously described.8 The isolated PRRSV samples from the lungs and lymph nodes were
further analyzed for ORF5 sequence.20 The methods were
previously approved by the Seoul National University, Institutional Animal Care and Use Committee.
Twenty pigs (10 pigs with each genotype of PRRSV)
each naturally infected with type 1 or 2 PRRSV from 1 pig
from each farm were also used for an evaluation of 2 mAbs
(SDOW17 and SR30)-based IHC. Four blocks of lung tissue from 8 different portions of 4 lobes (cranial, middle,
accessory, and caudal lobe) were collected, as previously
Journal
of
V e t e r i n a r y D i a g n o s t i c I n v e s t i g a t i o n 24 (2012)
described.12 The mAbs, SDOW17a and SR30a were diluted
1:10,000 in phosphate buffered saline (0.01 M, pH 7.4) containing 0.1% Tween 20. The IHC was performed as previously described.6
Probes used for the ISH assays were generated by RTPCR using genomic RNA of isolates SNUVR100057 and
SNUVR090851. For type 1 PRRSV, a 354-bp complementary (cDNA) fragment representing the 5’ region of
ORF6 and ORF7 was used as a probe. The forward and
reverse primers were 5’-CGCTGTGACAAAGCCCGG
AC-3’ (nucleotides 14,482–14,501) and 5’-TCGATTGCA
AGCAGAGGGAG-3’ (nucleotides 14,814–14,835), respectively. For type 2 PRRSV, a 349-bp cDNA fragmentrepresenting the 5’ region of ORF6 and ORF7 was used as a
probe. The forward and reverse primers were 5’-TCGTTC
GGCGTCCCGGCTCC-3’ (nucleotides 14,775–14,794) and
5’-TTGACGACAGACACAATTGC-3’ (nucleotides 15,122–
15,141), respectively.14 Polymerase chain reaction was carried out, as previously described.14 The purified PCR
product was labeled by random priming with digoxigenin–2’-deoxyuridine 5’-triphosphate (dUTP) with a commercial, kit.b The ISH was carried out as previously described.6 Reverse transcription nested PCR (RT-nPCR) was
also performed to detect type 1 and 2 PRRSV in the FFPE
lung tissues from the experimentally and naturally infected
pigs as previously described.9
To obtain quantitative data, analyses of slides was performed with the NIH Image J 1.43m Program.c For each
slide of lung tissue, 10 fields were randomly selected, and
the number of positive cells per unit area (0.95 mm2) was
counted, as previously described.13 The order of the serial
slides was the same for each test. The mean values were
also calculated.
Porcine reproductive and respiratory syndrome virus was
isolated from the lungs (30 pigs) of type 1 and 2 PRRSV-infected pigs at multiple dpi. The PRRSV isolated from all of
the infected pigs was confirmed by sequence analysis to be
the same propagating Korean type 1 and 2 PRRSV in the
challenge stock. No PRRSV was isolated from the lungs of
the negative-control pigs.
Type 1 PRRSV nucleic acid was detected in all 15 or 10
lungs of the pigs that were experimentally or naturally infected with type 1 PRRSV by RT-nPCR. Type 2 PRRSV
nucleic acid was detected in all 15 or 10 lungs of the pigs
that were experimentally or naturally infected with type 2
PRRSV by RT-nPCR.
Distinct positive immunohistochemical and hybridization signals by IHC and ISH, respectively, were detected
at each day postinoculation among the lungs of infected
pigs. Either type 1 or 2 PRRSV antigens or genomes were
detected in the lung from 7, 14, and 21 dpi by IHC and
ISH, respectively, in all of the infected pigs throughout
the experiment (Table 1). Positive cells typically exhibited
red reaction and dark-brown reaction product by IHC and
ISH, respectively, in the cytoplasm, without background
staining. The SDOW17- and SR30-based IHC detected
PRRSV in the pigs experimentally infected with type 1
and 2 PRRSV, respectively.
Detection
of
European
and
North American Porcine
reproductive and respiratory syndrome virus
721
Table 1. Comparison of Porcine reproductive and respiratory syndrome virus (PRRSV)–positive cells detected by immunohistochemistry (IHC) and in situ hybridization (ISH).
Days postinoculation
IHC
PRRSV Lobe Type 1 Type 2 Cranial Middle Accessory Caudal Cranial Middle Accessory Caudal SDOW17
7
7.8 ± 4.9 10.7 ± 5.3
11.3 ± 4.5
19.3 ± 4.1
13.5 ± 2.5
20.5 ± 7.1 27.3 ± 5.9 53.3 ± 4.8 14 4.5 ± 4.5 6.3 ± 4.1 8.8 ± 2.5 7.5 ± 4.7 8.8 ± 5.1 16.6 ± 6.8 22.3 ± 3.1 27.3 ± 5.4 21
1.8 ± 0.9 2.3 ± 1.3 2.5 ± 1.3 2.7 ± 1.3 5.6 ± 1.7 6.5 ± 3.5 7.5 ± 2.6 9.8 ± 5.3 SR30 7
14 11.8 ± 5.4 16.5 ± 8.1 18.8 ± 9.3† 25.5 ± 7.9 14.5 ± 8.5 24.5 ± 8.7 33.3 ± 6.7 54.3 ± 7.6 21
8.8 ± 4.8 4.5 ± 3.1 11.8 ± 2.2 3.8 ± 2.1 14.3 ± 5.3 4.3 ± 1.9 18.3 ± 7.2† 7.8 ± 4.9 9.5 ± 5.8 6.7 ± 3.1 21.3 ± 4.6 9.5 ± 3.7 27.5 ± 8.7 8.3 ± 2.8 30.3 ± 4.6 11.8 ± 5.3 ISH*
7
16.5 ± 9.3 21.5 ± 8.1 23.8 ± 4.1‡ 30.5 ± 7.9 21.8 ± 8.6 32.5 ± 8.4 38.3 ± 9.3 59.3 ± 5.3 14 21
11.3 ± 7.2 5.3 ± 2.9
15.8 ± 6.7‡ 7.5 ± 4.1
17.8 ± 6.3 5.3 ± 0.9
23.3 ± 7.2‡ 8.3 ± 6.1
12.3 ± 8.1 8.8 ± 1.7
25.5 ± 5.3 9.3 ± 2.8
32.5 ± 8.7 11.3 ± 3.3
35.3 ± 6.8 16.8 ± 5.3
* Type 1 and type 2 PRRSV nucleic acid was detected by type 1– and type 2–based ISH, respectively.
† Significantly different PRRSV-positive cells between SDOW17 and SR30 monoclonal antibody–based IHC at the same days postinoculation.
‡ Significantly different PRRSV-positive cells between SDOW17 monoclonal antibody–based IHC and type 1–based ISH at the same days
postinoculation.
The type 1 PRRSV-based ISH detected the type 1 PRRSV
from pigs experimentally infected with type 1 PRRSV. The
type 2 PRRSV-based ISH detected the type 2 PRRSV from
pigs experimentally infected with type 2 PRRSV. The type
1 PRRSV-based ISH does not detect the type 2 PRRSV from
pigs experimentally infected with type 2 PRRSV and vice
versa. The SDOW17- and SR30-based IHC and type 1 (or
type 2)–based ISH of the serial sections of lung indicated
that cells that stained positive for type 1 (or type 2) PRRSV
antigen were also positive for type 1 (or type 2) PRRSV nucleic acid (Figs. 1, 2).
The SR30-based IHC detected significantly more type
1 PRRSV-positive cells in the accessory and caudal lobes
from the experimentally infected pigs at 7 (P = 0.025) and
14 (P = 0.018) dpi, respectively, compared to the SDOW17based IHC. The type 1–based ISH detected significantly
more type 1 PRRSV-positive cells in the accessory lobe (P
= 0.007) at 7 dpi and the middle (P = 0.045) and caudal (P
= 0.018) lobes from the experimentally infected pigs at 14
dpi, as compared to the SDOW17-based IHC (Table 1).
SDOW17- and SR30-based IHC detected both type 1
and 2 PRRSV from the 20 pigs (10 pigs in each genotype
of PRRSV) naturally infected with type 1 and 2 PRRSV, respectively. The type 1 PRRSV-based ISH detected the type
1 PRRSV from pigs naturally infected with type 1 PRRSV.
The type 2 PRRSV-based ISH detected the type 2 PRRSV
from pigs naturally infected with type 2 PRRSV. There
were no significantly different type 1 and 2 PRRSVpositive
cells among the SDOW17- and SR30-based IHC and type 1
and 2–based ISH.
The results of the present study demonstrated that
IHC and ISH are of potential value to detect both type 1
and 2 PRRSV in FFPE lung tissues. Because the SDOW17
and SR30 mAbs recognize a highly conserved epitope on
the nucleocapsid (N) protein,19,25 these 2 mAbs are valuable diagnostic tools for the detection of 2 genotypes of
PRRSV. However, there are clear differences between IHC
and ISH for the detection and differentiation of the 2 genotypes of PRRSV in FFPE lung tissues from naturally and
experimentally infected pigs. Although mAb-based IHC
is specific for the detection of both 2 genotypes of PRRSV,
none have been shown to differentiate type 1 PRRSV from
type 2 PRRSV.
Reverse transcription nPCR alone cannot evaluate the severity of histopathological lesions induced by PRRSV, although RT-nPCR can differentiate between the 2 genotypes
of PRRS and can measure the amount of PRRSV.8,9 ISH can
differentiate between the 2 genotypes of PRRSV in FFPE tissues, as previously reported,15 and provides histological architecture, thus, the detection of the causative organism and
histopathological evaluation may be performed simultaneously in the same section using genotypespecific ISH. Routine use of ISH is largely restricted to diagnostic laboratories
because the technique is technically more complex, cumbersome, and expense when compared with IHC. However, the
ease of preparation of cDNA probes by RT-PCR compared
with the generation of either monoclonal or polyclonal antibodies, combined with the increasing availability of probes
and sequence databases, render the technique more applicable in diagnosis and research areas in the future.
The SR30 mAb–based IHC is more sensitive in the detection both type 1 and 2 PRRSV in FFPE lung tissues than
SDOW17 mAb–based IHC. Similar to ISH, IHC also provides cellular detail and histological architecture so that
thenumber of PRRSV-infected cells and lesions may be observed simultaneously in the same section. This advantage
is critical for the evaluation of the PRRSV strain in infected
pigs as a more virulent PRRSV strain can induce more se-
722 Han
et al. in
Figure 1. Serial section of a lung from a pig experimentally infected with type 1 Porcine reproductive and respiratory syndrome
virus (PRRSV) at 7 days postinoculation. A) type 1 PRRSV antigen was detected in the macrophages with a SDOW17 monoclonal antibody (mAb)–based immunohistochemistry. B)
type 1 PRRSV antigen was detected in the macrophages with
a SR30 mAb-based immunohistochemistry. C) type 1 PRRSV
nucleic acid was detected in the macrophages with a type 1–
based in situ hybridization.
Journal
of
V e t e r i n a r y D i a g n o s t i c I n v e s t i g a t i o n 24 (2012)
Figure 2. Serial section of a lung from a pig experimentally infected with type 2 Porcine reproductive and respiratory syndrome
virus (PRRSV) at 7 days postinoculation. A) type 2 PRRSV antigen was detected in the macrophages with a SDOW17 monoclonal antibody–based (mAb) immunohistochemistry. B)
type 2 PRRSV antigen was detected in the macrophages with
a SR30 mAb-based immunohistochemistry. C) type 2 PRRSV
nucleic acid was detected in the macrophages with a type 2–
based in situ hybridization.
Detection
of
European
and
North American Porcine
reproductive and respiratory syndrome virus
vere interstitial pneumonia histopathologically than a lessvirulent strain regardless of genotype of PRRSV.11,12
Presently, there is no ideal practical diagnostic method
that allows detection and differentiation of the 2 genotypes
of PRRSV in FFPE tissues. Based on the present results,
IHC is the practical diagnostic method. However, it will be
necessary to develop a genotype-specific PRRSV mAb to
differentiate between the 2 genotypes of PRRSV for IHC in
FFPE lung tissues.
Sources and manufacturers
a. Rural Technologies Inc., Brookings, SD.
b. Boehringer Mannheim, Indianapolis, IN.
c. http://rsbweb.nih.gov/ij
Acknowledgments — This research was supported by contract research funds of the Research Institute for Veterinary
Science (RIVS) from the College of Veterinary Medicine and
by the Brain Korea 21 Program for Veterinary Science in the
Republic of Korea.
References
1. Allende R, Lewis TL, Lu Z, et al.: 1999, North American and
European porcine reproductive and respiratory syndrome
viruses differ in non-structural protein coding regions. J
Gen Virol 80:307–315.
2. Amonsin A, Kedkovid R, Puranaveja S, et al.: 2009, Comparative analysis of complete nucleotide sequence of porcine
reproductive and respiratory syndrome virus (PRRSV) isolates in Thailand (US and EU genotypes). Virol J 6:143.
3. Beaver BV, Reed W, Leary S, et al.: 2001, 2000 report of the
AVMA panel on euthanasia. J Am Vet Med Assoc 218:669–
696. Erratum in J Am Vet Med Assoc 2001, 218:1884.
4. Cavanagh D: 1997, Nidovirales: a new order comprising
Coronaviridae and Arteriviridae. Arch Virol 142:629–633.
5. Chen N, Cao Z, Yu X, et al.: 2011, Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China. J Gen Virol 92:880–892.
6. Cheon DS, Chae C: 1999, Distribution of a Korean strain of
porcine reproductive and respiratory syndrome virus in
experimentally infected pigs, as demonstrated immunohistochemically and by in-situ hybridization. J Comp Pathol
120:79–88.
7. Cheon DS, Chae C: 2000, Antigenic variation and genotype
of isolates of porcine reproductive and respiratory syndrome virus in Korea. Vet Rec 147:215–218.
8. Cheon DS, Chae C: 2000, Comparison of virus isolation, reverse transcription-polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection
of porcine reproductive and respiratory syndrome virus
from naturally aborted fetuses and stillborn piglets. J Vet
Diagn Invest 12:582–587.
9. Chung HK, Choi C, Kim J, Chae C: 2002, Detection and differentiation of North American and European genotypes
of porcine reproductive and respiratory syndrome virus in
723
formalin-fixed, paraffin-embedded tissue by multiplex reverse transcription-nested polymerase chain reaction. J Vet
Diagn Invest 14:56–60.
10. Gagnon CA, del Castillo JR, Music N, et al.: 2008, Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an
epidemiological survey. J Vet Diagn Invest 20:545–558.
11. Halbur PG, Paul PS, Frey ML, et al.: 1995, Comparison of
the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad
virus. Vet Pathol 32:648–660.
12. Halbur PG, Paul PS, Frey ML, et al.: 1996, Comparison of
the antigen distribution of two US porcine reproductive
and respiratory syndrome virus isolates with that of the
Lelystad virus. Vet Pathol 33:159–170.
13. Jung K, Ha Y, Chae C: 2005, Pathogenesis of swine influenza virus subtype H1N2 infection in pigs. J Comp Pathol
132:179– 184.
14. Kono Y, Kanno T, Shimizu M, et al.: 1996, Nested PCR for detection and typing of porcine reproductive and respiratory
syndrome (PRRS) virus in pigs. J Vet Med Sci 58: 941–946.
15. Larochelle R, Magar R: 1997, Differentiation of North
American and European porcine reproductive and respiratory syndrome virus genotypes by in situ hybridization. J
Virol Methods 68:161–168.
16. Lee C, Kim H, Kang B, et al.: 2010, Prevalence and phylogenetic analysis of the isolated type I porcine reproductive
and respiratory syndrome virus from 2007 to 2008 in Korea. Virus Genes 40:225–230.
17. Mengeling WL, Lager KM: 2000, A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome. Vet
Res 31:61–69.
18. Nelsen CJ, Murtaugh MP, Faaberg KS: 1999, Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J Virol 73:270–280.
19. Nelson EA, Christopher-Hennings J, Drew T, et al.: 1993,
Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal
antibodies. J Clin Microbiol 31:3184–3189.
20. Oleksiewicz MB, Bøtner A, Madsen KG, Storgaard T: 1998,
Sensitive detection and typing of porcine reproductive and
respiratory syndrome virus by RT-PCR amplification of
whole viral genes. Vet Microbiol 64:7–22.
21. Ropp SL, Wees CE, Fang Y, et al.: 2004, Characterization of
emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States. J Virol
78: 3684–3703.
22. Thanawongnuwech R, Amonsin A, Tatsanakit A, Damrongwatanapokin S: 2004, Genetics and geographical variation of porcine reproductive and respiratory syndrome
virus (PRRSV) in Thailand. Vet Microbiol 101:9–21.
23. van Woensel PA, Liefkens K, Demaret S: 1998, Effect on viraemia of an American and a European serotype PRRSV
vaccine after challenge with European wild-type strains of
the virus. Vet Rec 142:510–512.
724 Han
et al. in
24. Wasilk A, Callahan JD, Christopher-Hennings J, et al.:
2004, Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and
relative quantitation in boar semen and serum samples by
real-time PCR. J Clin Microbiol 42:4453–4461.
25. Wootton SK, Nelson EA, Yoo D: 1998, Antigenic structure of the nucleocapsid protein of porcine reproductive
and respiratory syndrome virus. Clin Diagn Lab Immunol
5:773–779.
Journal
of
V e t e r i n a r y D i a g n o s t i c I n v e s t i g a t i o n 24 (2012)
26. Yaeger MJ: 2002, The diagnostic sensitivity of immunohistochemistry for the detection of porcine reproductive and
respiratory syndrome virus in the lung of vaccinated and
unvaccinated swine. J Vet Diagn Invest 14:15–19.
27. Zimmerman J, Benfield DA, Murtaugh MP, et al.: 2006,
Porcine reproductive and respiratory syndrome virus (porcine arterivirus). In: Disease of swine, ed. Straw BE, 9th ed.,
pp. 387–417. Blackwell, Ames, IA.