PCR-amplified 719 base pair bp cDNA fragments from porcine reproductive and respiratory syndrome virus PRRSV and their corresponding lung specimens digested with the restriction enzymes [r]
(1)NOTE Virology Polymerase Chain Reaction-Based Restriction Fragment Length Polymorphism Pattern of Porcine Reproductive and Respiratory Syndrome Virus Directly from Lung Tissues without Virus Isolation in Korea Doo-Sung CHEON1) and Chanhee CHAE1)* 1) Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441–744, Kyounggi-Do, Republic of Korea (Received 23 October 2000/Accepted 22 January 2001) ABSTRACT Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis was developed for directly typing porcine reproductive and respiratory syndrome virus (PRRSV) from lung specimens without virus isolation Twenty nine lung specimens collected from postweaning pigs were isolated for PRRSV When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig The results suggest that the PCR-based RFLP analysis method may be useful to distinguish PRRSV isolates directly from lung specimens without virus isolation KEY WORDS: polymerase chain reaction, porcine reproductive and respiratory syndrome virus, restriction fragment length polymorphism J Vet Med Sci 63(5): 567–571, 2001 Porcine reproductive and respiratory syndrome virus (RRRSV) is a member of the genus Arterivirus, family Arteriviridae, order Nidovirales and has a positive singlestranded polyadenylated RNA molecule of approximately 15 kb in length that contains eight open reading frames (ORFs) [3, 16] The first two open reading frames (ORF 1a and 1b) encode the viral RNA polymerase and associated proteases [7, 16] ORFs 2, 3, and encode structural proteins, ORF encodes a matrix (M) protein of 18 to 19-kD and ORF encodes a nucleocapsid (N) protein of 14 to 15kD [14, 16–18] The disease is characterized by reproductive failure through abortion, occurrence of stillbirth, and mummified fetuses and severe respiratory disease in newborn and nursing pigs [9] PRRSV apparently replicates primarily in interstitial and alveolar macrophages in the lung, and macrophage lineages in several other tissues such as tonsil, Peyer’s patch, liver and adrenal gland [4, 5, 10, 20] A procedure called polymerase chain reaction (PCR) can amplify a few copies of specific target DNA sequence within only a few hours Restriction fragment length polymorphism (RFLP) analysis is useful in molecular biology for differentiating DNA variation A PCR-based RFLP analysis method has been developed for differentiation of PRRSV isolates [8, 21] A recommended standardized method for RFLP analysis of PRRSV isolates includes digestion of PCR-amplified DNA from ORF with restriction enzymes [21] Previous data showed that PCR-based RFLP analysis was a rapid and sensitive method, but these studies were limited to PRRSV field strains isolated from lung or other specimens Here, we report a PCR-based RFLP analysis method * CORRESPONDENCE TO: DR CHAE, C., Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Suwon 441–744, Kyounggi-Do, Republic of Korea developed to differentiate PRRSV strains directly from lung specimens without virus isolation To validate this approach, we compared the RFLP patterns of PRRSV field isolates with those of their corresponding lung tissues derived from 29 pigs A total of 45 postweaning pigs, which were highly suggestive of PRRSV infection, were selected at necropsy from pigs submitted to the Department of Veterinary Pathology for diagnosing respiratory disease on the based of clinical sign (severe dyspnea) and gross lesions (interstitial pneumonia) Antibiotic therapy did not show any beneficial results in all 45 postweaning pigs Lung tissues were obtained from pigs which are submitted to Department of Veterinary Pathology, Seoul National University to diagnose respiratory disease Lung tissues were collected from similar locations where interstitial pneumonia was seen The original isolation of the Korean isolates was made on both MARC-145 cells and alveolar macrophages as previously described [12, 19, 22] The PRRSV vaccine strain VR2332 was used as the reference strain [6] PRRSV isolates were propagated on MARC-145 cells The MARC-145 cells were cultured at Eagle’s minimum essential medium, supplemented with 8% fetal bovine serum in humidified 5% CO2 atmosphere at 37°C PRRSV field isolates were harvested when 70% to 80% of the cells were affected by cytopathic changes RNA was extracted from infected cells with the Trizol LS reagent (Gibco BRL, Grand Island, NY, U.S.A.) according to the manufacturer’s instructions Pieces of approximately gram were ground with 20 ml of Eagle’s minimum essential medium by homogenizer Homogenized lung samples were centrifuged for 20 at 5,000 × g and the supernatants were transferred into a fresh tube The 500 µl of supernatants were mixed with equal volume of Trizol LS (Gibco BRL) and then incu- (2) 568 D.-S CHEON AND C CHAE bated for 10 at room temperature After adding 200 µl of CCl4 and mixing by vortexing the tube, the sample was incubated for 15 at room temperature Then it was centrifuged for 15 at 3,500 × g and the aqueous phase were removed Equal volume of cold isopropanol was added, mixed immediately by vortexing the tube, and incubated for overnight at –20°C The precipitated RNA was pelleted by centrifugation for 10 at 14,000 × g Twenty-five picomoles of oligo(dT) was added to µg of extracted RNA from either PRRSV-infected MARC-145 cells or lung specimens in µl of diethyl pyrocarbonate (DEPC; Sigma Chemical Co., St Louis, MO, U.S.A.)treated water, denatured at 75°C for 10 min, and then cooled on ice Reverse transcription reaction was performed in a final volume of 20 µl containing × RT buffer (50 mM TrisHCl, [pH 8.3], 75 mM KCl, mM MgCl2), 0.5 mM (each) deoxynucleotide triphosphates (dNTP), 20 U of RNase inhibitor per µl, and 50 U of murine leukemia virus reverse transcriptase (Perkin Elmer, Foster City, CA, U.S.A.) After incubation for hr at 42°C, the mixture was heated at 95°C for and chilled on ice The two 20-base oligonucleotides used as primers for the PCR assays were selected from published sequences of ORF The forward and reverse primers were 5’-CCATTCTGTTGGCAATTTGA-3’ and 5’-GGCATATATCATCACTGGCG-3’, respectively [1] cDNA amplification was performed using 25 pmole oligonucleotide primers, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, 10 mM Tris HCl (pH 8.8); 1.25 mM MgCl2; 50 mM KCl; 1.25 unit of Taq polymerase, all in 50 µl of distilled water The PCR profile used included a denaturing step at 94°C for 30 sec, followed by annealing of the primers at 53°C for 30 sec, with an extension step at 72°C for 30 sec The thirty cycles of this 3-step procedures were performed in a thermal cycler, followed by a 10 extension at 72°C The sensitivity of detection of PRRSV with primer set was tested A dilution series of tissue culture-grown virus was made in culture medium From each dilution, nucleic acid was extracted for cDNA synthesis and an equivalent aliquot was taken for tissue culture infective does 50 (TCID50) assay, which were set up on the same day Complementary DNA synthesis was followed by RT-PCR Thus, the end point of detection of infectious PRRSV could be directly compared with the end point of detection of viral RNA by RT-PCR After amplification, the PCR products were purified with Wizard PCR Preps (Promega Biotech, Madison, WI, U.S.A.) The amplified cDNA was then digested with the restriction enzyme MluI, HincII, SacII, and HaeIII (Promega) in the appropriate buffer solution for hr at 37°C as previousely described except HaeIII [21] The digested cDNA fragments were analyzed by electrophoresis on 2.0% agarose gels (MetaPhor agarose; FMC Products, Rockland, ME, U.S.A.) The gels were examined by transillumination and photographied The size of digested cDNA fragments was estimated from migration distances of molecular weight standards Twenty nine of 45 dyspneic postweaning pigs were positive for RT-PCR and virus isolation Four cases were positive for RT-PCR only Twelve cases were negative for both RT-PCR and virus isolation Therefore, a total of 29 dyspneic postweaning pigs which were positive for both RTPCR and virus isolation were used in this study Among 29 PRRSV isolates, was isolated in 1996, 13 were isolated in 1997, and 15 were isolated in 1998 Eighteen were isolated in Kyounggi Province, were isolated in Kyoungsang Province, were isolated in Chungcheung Province, and were isolated in Cholla Province (Table 1) No commercial PRRSV vaccine had been used in any herds from which PRRSV was isolated All 29 PRRSV isolates showed reactivity to anti-PRRSV monoclonal antibody SDOW17 The focus of PRRSV-infected MARC-145 cells shows an intense, often granular cytoplasmic fluorescence observed days postinoculation When cDNAs prepared from the 29 PRRSV isolates and the corresponding lung specimens, from which the PRRSV isolates were isolated, were used as template, the PCR assay yielded a fragment of the expected 716 base pair (bp) of the same molecular weights as those obtained with the cDNA preparation from PRRSV strain VR2332 and SNUVR970501 (Fig 1) Ten of those PCR products were sequenced, and their identity was confirmed as ORF of PRRSV (data not shown) The RT-PCR was able to detect 10 TCID50/ml of PRRSV with primer pair The end point of detection for each virus remained an apparent amplification band In order to determine whether the PCR-based RFLP analysis method could differentiate PRRSV field viruses directly lung specimens without virus isolation, the PCR assay was performed on cDNA preparations from the 29 PRRSV field viruses and from their corresponding lung specimens The 716 bp fragments produced by PCR assay were further digested with four restriction enzymes, MluI, HincII, SacII, HaeIII, and analyzed by agarose gel electrophoresis When the PCR products from the 29 PRRSV field viruses were subjected to RFLP analysis, it showed that 2, 6, and distinguishable digestion patterns were generated by MluI, HincII, SacII and HaeIII, respectively To facilitate the reporting and handling of test data, each isolate and lung specimen was given a numerical code for its ORF RFLP pattern with enzymes, MluI, HincII, SacII, HaeIII, in that order (Table 1) Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, HaeIII, we observed 15 distinct RFLP patterns from the 29 PRRSV field viruses tested (Table 1) Representative results of restriction enzyme digestion of PCR-amplified products from PRRSV isolates and their corresponding lung specimens from each pig are shown in Fig When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig Nineteen (66%) out of 29 field viruses had an RFLP pattern for ORF identical or similar to attentuated live (3) 569 RFLP ANALYSIS OF PRRSV Table Comparison of 29 PRRSV field viruses and their corresponding lung specimens by polymerase chain reaction-based restriction fragment length polymorphism (RFLP) analysis RFLP codea) PRRSV No of field virus Passage Isolate Lung Isolated Year 961170 970501 970686 970758 971087 971224 971248 971477 971506 971510 971511 971512 971546 971547 980099 980143 980164 980167 980168 980210 980261 980262 980353 980386 980397 980501 980515 980559 980567 VR2332 3 3 4 3 3 2 2321 2321 2323 2323 2323 1232 2331 2333 1331 2321 1416 1416 2321 2321 2321 2321 2521 1424 1424 2321 2121 2131 2321 1631 2321 1231 1412 1631 2321 2321 2321 2321 2323 2323 2323 1232 2331 2333 1331 2321 1416 1416 2321 2321 2321 2321 2521 1424 1424 2321 2121 2131 2321 1631 2321 1231 1412 1631 2321 NAb) 1996 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 Province Kyounggi Kyounggi Kyoungsang Kyounggi Chungcheung Kyounggi Kyoungsang Kyounggi Kyounggi Kyoungsang Kyounggi Kyounggi Cholla Kyounggi Kyounggi Kyounggi Kyoungsang Kyoungsang Kyounggi Cholla Kyounggi Chungcheung Kyoungsang Kyounggi Kyounggi Kyounggi Kyounggi Kyoungsang Kyounggi a) Each isolate and lung specimen was given a numerical code for its ORF RFLP pattern with enzymes, MluI, HincII, SacII and HaeIII, in that order b) Not available Fig Representative results for PCR-amplified 719 base pair (bp) cDNA fragments from porcine reproductive and respiratory syndrome virus (PRRSV) isolates and their corresponding lung specimens Lane M is a 100 bp ladder Lane is a PRRSV VR2332 strain Lanes 2, 4, 6, and 10 are PRRSV isolates Lanes 3, 5, 7, and 11 are the corresponding lung specimens of the proceeding even-numbered lane, respectively PRRSV strain VR2332 Among the 19 field viruses, 12 were identical RFLP code 2321 Field viruses with RFLP code 2321 were isolated from postweaning pigs from 1996 to 1998 throughout the country Ten field viruses had different RFLP pattern for ORF from attenuated live PRRSV strain VR2332 (Table 1) We have established a new method to sensitively, accurately, and rapidly detect and identify PRRSV from lung specimens by PCR-based RFLP analysis without virus isolation This method could completely differentiate PRRSV isolates Virus isolation is time-consuming and not very sensitive The failure of virus isolation has made epidemiological surveillance and management of outbreak more difficult The primary advantage of the PCR-based RFLP method described here is that viral cDNA can be amplified directly from lung specimens without virus isolation and results can be obtained within 24 hr Several PCR-based RFLP analysis protocols have been proposed for the differentiation of PRRSV strains from field isolates [8, 21] Primers pairs for detecting and typing PRRSV, based on amplifying ORF 5, and genes, have (4) 570 D.-S CHEON AND C CHAE Fig PCR-amplified 719 base pair (bp) cDNA fragments from porcine reproductive and respiratory syndrome virus (PRRSV) and their corresponding lung specimens digested with the restriction enzymes to MluI (a), HincII (b), SacII (c) and HaeIII (d) The left lane of each pair is a PRRSV isolate and the right lane is the corresponding lung specimen Lane M is a 100 bp DNA ladder been described [8, 21] These results have demonstrated a great diversity of ORF 5, and among PRRSV field isolates These diversities of ORF by PCR-based RFLP were confirmed by animo acid sequence analysis The amino acid sequences of ORFs to of PRRSV vary widely with those derived from the nucleotide sequences of European and North American isolates [11, 18] Amplification of ORF was chosen for PCR-based RFLP analysis, because it has already been reported in ORF 5-dervied PCR-based RFLP analysis of PRRSV isolated from other country [8, 21] and extensive DNA sequence information is available for this region [11, 16, 21] Therefore, it is possible to compare the RFLP patterns of PRRSV with those from other countries The PRRSV vaccine started to be used in Korea since 1996 The relationship between the live vaccine strain and the PRRSV prevalence is not determined because no differential serologic test distinguishes vaccinated animals from field virus-infected pigs The high prevalent of field viruses that had an RFLP pattern for ORF identical or similar to attenuated live PRRSV vaccine strain VR2332 but not to the prototype VR2332 is surprising In the United States, none of the field viruses isolated before use of the attenuated live PRRSV vaccine had an RFLP pattern for ORF identical or similar to vaccine strain [11, 14, 21] Field viruses that had an RFLP pattern for ORF identical or similar to vaccine strain were also reported in United States and Denmark after use of vaccine [2, 13, 15] Notably, whereas the vaccine strain did not cause detectable pathologic changes other than some lymph node enlargement, field strains that had an RFLP pattern for ORF identical or similar to vaccine strain were associated with pulmonary lesions [15] All field viruses that had an RFLP pattern for ORF identical or similar to vaccine strain were isolated from the field cases associated with typical PRRS in the present study Further studies are needed to determine the vaccine strain may have reverted to a more virulent virus PCR-based RFLP analysis of typing PRRSV isolates directly from lung specimens without virus isolation is sensitive, accurate, and rapid The results presented here suggest that the protocol may be useful for epidemiologic studies of PRRSV infection and for monitoring herds before and after eradication to determine whether recurrence of PRRS following PRRSV eradication is due to recrudescence or 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