Molecular characterization of recent Korean porcine reproductive and respiratory syndrome (PRRS) viruses and comparison to other Asian PRRS viruses

Veterinary Microbiology 117 (2006) 248–257 www.elsevier.com/locate/vetmic

Short communication

Molecular characterization of recent Korean porcine reproductive and respiratory syndrome (PRRS) viruses and comparison to other Asian PRRS viruses Sang-Ho Cha a,b, Eun-Jin Choi a,d, Jong-Hyeon Park a, So-Rah Yoon a, Jae-Young Song a, Jun-Hun Kwon a, Hee-Jong Song d, Kyoung-Jin Yoon b,c,* a

Division of Virology, National Veterinary Research and Quarantine Service, Anyang, Gyeonggido, Republic of Korea b Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA c Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA d College of Veterinary Medicine, Jeon-Buk National University, Republic of Korea Received 21 December 2005; received in revised form 6 May 2006; accepted 16 May 2006

Abstract Twenty-eight PRRS viruses (PRRSVs) isolated from various pig farms in Korea between 2002 and 2003 were sequenced for open-reading frame (ORF) 5 and/or full-length genome and compared with numerous PRRSVs reported from North America, Europe and Asia. All Korean isolates examined were genetically of the North American genotype. The ORF5 sequence of one isolate was identical to Ingelvac1 PRRS MLV vaccine virus. ORF5 nucleotide sequence divergence of the remaining 27 Korean PRRSVs from VR-2332, the prototype of the North American PRRSVand parental strain of the MLV vaccine virus, ranged from 1.3% to 12.9%, which corresponded to 2.0% to 14.9% divergence at the amino acid level, raising a concern on the efficacy of the MLV vaccine. Phylogenetic analyses of ORF5 and/or full-length sequences revealed that the Korean PRRSVs formed a clade distinct from PRRSVs reported from other Asian countries (China, Taiwan, Japan, and Thailand). Our study demonstrated that PRRSVs of the North American genotype were introduced to the Korean swine population some time ago and have evolved independently from PRRSV in other Asian countries, suggesting that geographic separation might influence the molecular evolution of PRRSV. This should be taken into consideration when a national PRRS prevention and control policy for international trade is established. # 2006 Elsevier B.V. All rights reserved. Keywords: PRRSV; Korea; ORF5; Full-length genome; Phylogenetic analysis; Asian countries

* Corresponding author at: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA. Tel.: +1 515 294 1083; fax: +1 515 294 6619. E-mail address: [email protected] (K.-J. Yoon). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.05.007

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1. Introduction Porcine reproductive and respiratory syndrome (PRRS) has been reported in pig producing regions throughout the world (Zimmerman and Yoon, 2003). The syndrome is caused by PRRS virus (PRRSV) which is a member of the family Arteriviridae in the order of Nidovirales (Cavanagh, 1997). The virus is enveloped and contains a polyadenylated, singlestranded, positive-sense RNA genome of 15 kb in

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length which consists of nine open-reading frames (ORFs) (Snijder and Meulenberg, 1998; Wu et al., 2001). ORF5 that encodes for the major envelope protein of the virus has been the target gene for molecular epidemiology of PRRS viruses (PRRSVs) by sequence analysis because ORF5 has shown the highest genetic variability among PRRSVs (Key et al., 2001). PRRSVs can be divided into European (type I) and North American (type II) genotypes, which are represented by Lelystad and VR-2332 strains,

Fig. 1. Geographic origins of Korean PRRSV isolates used in the study.

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respectively. Two genotypes share less than 60% nucleotide identity (Nelsen et al., 1999) and are distinguished not only genetically but also antigenically (Meng, 2000). Genetic variation among PRRSVs within each genotype also has been reported and viruses tend to be clustered by geographical origin (Forsberg et al., 2002; Larochelle et al., 2003; Mateu et al., 2003; Thanawongnuwech et al., 2004). In Asia, PRRS outbreaks were reported in many countries between the late 1980s and early 1990s (Zimmerman and Yoon, 2003). Genetic analyses have shown that most of the PRRSVs identified in the People’s Republic of China, Japan, Taiwan and Thailand are of type II PRRSV (Chueh et al., 1998; Damrongwatanapokin et al., 1996; Jiang et al., 2000; Yoshii et al., 2004). A recent study, however, demonstrated the presence and higher prevalence of type I PRRSV in Thailand (Thanawongnuwech et al., 2004). Introduction of PRRSV into the Republic of Korea was dated back as early as 1985 by a serological survey

(Shin et al., 1993). However, the first successful isolation of PRRSV, which was antigenically similar to contemporary U.S. PRRSVs at that time, could not be made until 1993 (Kweon et al., 1994). The subsequent molecular characterization of PRRSVs collected between 1996 and 1998 also revealed that the Korean viruses belonged to type II (Cheon and Chae, 2000). The following study was conducted to genetically characterize more contemporary Korean PRRSVs (2002 and 2003) in comparison to Asian, North American and European PRRSVs as part of the development of a national PRRS control strategy and policy.

2. Materials and methods 2.1. Study design Tissues and serum samples were collected from 97 commercial pig farms in various geographical regions

Table 1 Primers used for amplification and sequencing of PRRS viral genome Fragment

Primer

Sequence 0

Location 0

1

F1F F1R

5 -ATGACGTATAGGTGTTGG-3 50 -GCGGATCCAACTCTCCTTAACGG-30

1–18 1181–1203

2

F2F F2R

50 -CTAAACGGACCTATCGTCG-30 50 -AGGTGTCGATTACGCGTGCG-30

1150–1168 2172–2191

3

F3F F3R

50 -GATTGACCTGTACCTCCGTGG-30 50 -CTGCTTGATGACACGGACG-30

2103–2123 3194–3212

4

F4F F4R

50 -GCATGAAGCTGAGGAAACC-30 50 -ATGGAACAGCGGAAACCTTGACC-30

3135–3153 4719–4741

5

F5F F5R

50 -CTGTATCTTGGCTGGAGCTTACGTGC-30 50 -GCATGTCCCATCATTCTCCACAGG-30

4329–4354 6270–6293

6

F6F F6R

50 -CTTTGTGCCTTGCTTGCTGCC-30 50 -CTTTGGCAGTCAGTTCGC-30

6193–6213 7613–7630

7

F7F F7R

50 -CTCTGTGTGGACATGTCACCATTGA-30 50 -GCGAGTCTGTTATTGCTGTC-30

7451–7475 9314–9333

8

F8F F8R

50 -CTGCTGCCACGACTTACTGG-30 50 -GAGAATCTACAACACGCTTGTCTGT-30

8981–9000 10890–10914

9

F9F F9R

50 -CATTCGATGTGGTTACATTGCATTTGCCC-30 50 -CCAACCGGCGATGGTGAAGC-30

10630–10658 12190–12209

10

F10F F10R

50 -ATGAAATGGGGTCCATGCA-30 50 -TGCTGCTTGCCGTTGTTA-30

12073–12091 14897–14914

11

F11F FUR

50 -GTCGTCCTTAGATGACTTCTGTCAT-30 50 -AATTTCGGCCGCATGGTTCTCGCCA-30

14380–14404 15387–15411

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Fig. 2. Phylogenetic relationship of Korean PRRS viruses within themselves and to PRRS viruses of North American genotypes reported in various countries based on ORF 5 sequence. Reliability of the tree was assessed by bootstrap analysis with 1000 replications.

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of Korea undergoing reproductive and/or respiratory problems between 2002 and 2003. All samples were tested by RT-PCR assays designed to detect type I or II PRRSV (Gilbert et al., 1997; Oleksiewicz et al., 1998; Chang et al., 2002). PCR-positive samples were then subjected to virus isolation test. Because no type I virus was detected by PCR, virus isolation was attempted using an African monkey kidney cell line MARC-145 (Kim et al., 1993). Selected virus isolates were sequenced for ORF5 or complete genome and compared to each other and PRRSVs previously reported in various countries. 2.2. Viruses Twenty-eight PRRSVs each of which represented a separate swineherd in different geographic regions of Korea (Fig. 1) were selected and used in the study (Table 1). For comparison purpose, ORF5 sequences of 14 PRRSV isolates (GenBank accession nos. AY318766, AY318774, DQ480116–DQ480127) from U.S. Midwest swine population between 2002 and 2003 were used in this study. Lelystad (M96262) and VR-2332 (U87392) were used as the prototype reference viruses for types I and II, respectively. Ingelvac1 PRRS MLV (hereafter MLV) vaccine virus (AF066183) was also included in the comparison since this vaccine was licensed in Korea. In addition, many PRRSVs reported from North America, Europe and Asia whose sequences were available from GenBank were also used for comparison. 2.3. ORF5 RT-PCR and sequencing Total RNA was extracted from each sample by using the QIAamp1 Viral RNA mini kit (Qiagen Inc., Valencia, CA, USA) by following the manufacturer’s protocol. The entire ORF5 was amplified from the RNA extract by RT-PCR using the OneStep RT-PCR kit (Qiagen) and two primers: PR5F (50 -CCA TTC TGG TGG CAA TTT GA-30 ) and PR5R (50 -GGC ATA TAT CAT CAC TGG CG-30 ). The primers were designed based on the sequence of VR-2332. Reverse transcription and PCR amplification were performed as previously described (Cha et al., 2004). PCR products (715 bp), including complete ORF5 and the flanking region of ORFs 4 and 6, were purified with QIA quick1 purification kit (Qiagen) and then sequenced

with PR5F and PR5R primers (5 pmol each) using 377 Sequencer (PE Applied Biosystems, Foster City, CA, USA). 2.4. Full-length sequencing Twenty-two primers (Table 1) were designed to amplify 11 fragments of the full-length viral genome based on sequence of VR-2332 and a previous report (Wootton et al., 2000). RT-PCR was performed with 100 U Superscript1 III reverse transcriptase (Invitrogen, Grand Island, NY, USA) for RT and GeneAmp1 XL PCR kit (PE Applied Biosystems) for PCR amplification in the GeneAmp1 PCR system 2400 (PE Biosystems). Each of the amplified genes was then purified with QIAquick1 gel extraction kit (Qiagen) and cloned into T vector (Promega, Madison, WI, USA) for sequencing by following the manufacturer’s protocol. The Primer Walk sequencing was performed in both directions using the Big Dye1 Terminator cycle sequencing kit (PE Applied Biosystems). Untranslated region (UTR) at 50 and 30 ends of the viral genome was amplified with 50 and 30 RACE system (Invitrogen) for rapid amplification of cDNA ends by following the manufacturer’s instructions. All amplified genes were purified and then cloned into T vector (Promega) for sequencing. 2.5. Data analysis Multiple sequence alignment was carried out using sequence analysis software, Lasergene1 (DNASTAR Inc., Madison, WI, USA) to determine sequence homology and phylogenetic relationship among the viruses. Unrooted phylogenetic trees were generated by the distance-based neighborjoining method using MEGA software (Version 3.1). Bootstrap values were calculated on 1000 replicates of the alignment.

3. Results 3.1. PCR-based surveillance of PRRSV genotype No type I PRRS viral RNA was detected in any of the clinical specimens tested. In contrast, type II PRRS

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viral RNA was detected in serum and/or tissue samples from 77 of the 97 pig farms examined. 3.2. Phylogenetic analyses of ORF5 of Korean PRRS viruses All 28 isolates (DQ473448–DQ473474) had 603 nucleotides in ORF5 except for one isolate designated as JTS30 that had an in-frame deletion of three nucleotides corresponding to the amino acid position 34. The nucleotide (amino acid) divergence of ORF5 among the Korean PRRSV isolates ranged from 0.5% (1.0%) to 15.0% (16.8%). One isolate (KTS) showed 100% homology with the MLV vaccine virus. Sequence divergence of the remaining 27 viruses from VR-2332 was 1.3–12.9% and 2.0–14.9% at the nucleotide (NT) and amino acid (AA) levels, respectively. Similarly, the NT (AA) divergence of fourteen 2002–2003 U.S. PRRSVs from the VR-2332 strain was 0.5–16.6% (1.5–18.7%). The NT (AA) divergence of ORF5 between the 2002 and 2003 Korean PRRSVs and the 2002–2003 US PRRSVs

253

ranged from 0.2% (0.5%) to 18.3% (19.3%). In contrast, the NT (AA) divergence from European strains was 34.8–40.1% (42.8–50.7%). Phylogenetically, the ORF5 sequences of the Korean isolates were of type II. While four Korean isolates (KTS, DO47-2, 224-1, and MJH14) were classified into the VR-2332-like group, the remaining 24 isolates formed four different subgroups based on phylogenetic distance from the VR-2332-like group, with one being the closest to the group (Fig. 2). Although many of AA changes were found in the reported linear epitopes (Rodriguez et al., 2001; Ostrowski et al., 2002; Plagemann et al., 2002) and hypervariable regions of ORF5 (Key et al., 2001) among viruses representing each of the subgroups identified (Fig. 3), no significant correlation was observed between the isolation year of the viruses (i.e., chronological order) and phylogenetic distance of each subgroup from VR-2332. Relative to type II PRRSVs reported in Asian countries, the Korean isolates formed their own clade separated from PRRSVs originated in China, Japan,

Fig. 3. Multiple alignments of ORF5 amino acid sequence of PRRS viruses identified in various Asian countries. The dot-line box indicates Korean isolates representing each of four phylogenetic subgroups. The solid-line boxes represent linear epitopes (28–30 and 170–200) and a neutralizing epitope (37–45). Hypervariable regions of ORF5 are indicated by highlighting amino acid sequence of VR-2332 with gray color.

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Taiwan and Thailand (Fig. 2). Type II Chinese PRRSVs were genetically closer to JA142 (AY424271), the parental strain of the Ingelvac1 ATP vaccine virus, whereas Thai PRRSVs (2000–2002) were closer to IAF-exp91, a Canadian PRRSV (L40898). Japanese and Taiwanese PRRSVs appeared to be genetically related to each other based on the ORF5 sequence. Among the 14 2002–2003 U.S. isolates, two (02-61079 and 03-12476) belonged to the VR-2332-like group and one (A1) was close to Taiwanese isolate WSV (AF121267). However, the rest of the viruses formed their own clades independent of the Asian isolates. 3.3. Analysis of full-length genomic sequence of LMY strain A Korean PRRSV isolate designated LMY was selected for full-length sequencing because the virus showed the most divergence in the ORF5 region when it was compared with other Korean PRRSVs isolated in 2002. The genomic RNA of the LMY strain was 15,411 bp in length and had 32 poly(A) tail

(DQ473474). The genome was composed of nine ORFs (1a, 1b, 2, 2b, and 3–7). No additions or deletions were observed in the genomic sequence of LMY when compared with that of VR-2332. The 50 UTR of the LMY strain consisted of 189 nucleotides and showed 4.4% divergence from that of PL97-1 (AY585241) (Kang et al., 2004) and VR-2332. The 30 UTR consisted of 151 nucleotides and was divergent 8.4–8.5% from that of PL97-1 and VR2332. Leader-body junction sequence (TTAACC) in the leader sequence of the LMY strain was conserved when compared with PL97-1, VR-2332 or MLV. The ORF1a of the LMY strain encoded a polyprotein of 2503 amino acids, producing potentially nine cleavage products. The NT (AA) sequence divergence of each postulated ORF1a cleavage protein in comparison to VR-2332 ranged from 2.1% (0%) to 12.7% (14.3%). While no AA change was observed in Nsp6 and Nsp8 when compared to VR-2332, the highest sequence difference was detected in Nsp1b. ORF1b was expected to produce four cleavage proteins (Nsp9–Nsp12) from a polyprotein of 1457

Table 2 Nucleotide (NT) and deduced amino acid (AA) sequence divergence of 2002 Korean PRRSV isolate (LMY) from 1997 Korean PRRSV isolate (PL97-1), 1990 prototype North American PRRSV isolate (VR-2332) and 1990 prototype European PRRSV isolate (Lelystad) ORF or region

Protein product

PL97-1 NT (%)

0

5 UTR ORF1a

VR-2332 AA (%)

NT (%)

Lelystad AA (%)

NT (%)

AA (%)

Nsp1a Nsp1b Nsp2 Nsp3 Nsp4 Nsp5 Nsp6 Nsp7 Nsp8

4.4 4.4 9.6 7.6 6.1 5.1 6.6 2.1 3.2 3.0

1.8 12.6 10.4 3.4 3.5 5.5 0.0 2.8 0.0

4.4 4.2 9.9 7.3 6.1 5.0 6.4 2.1 3.0 2.3

1.8 14.3 9.9 3.4 3.0 6.1 0.0 2.4 0.0

49.8 36.7 49.1 53.3 40.6 40.2 35.1 27.1 48.6 39.3

33.1 64.8 73.1 42.4 40.4 29.4 18.8 57.9 31.8

ORF1b

Nsp9 Nsp10 Nsp11 Nsp12

3.3 2.5 7.5 12.4

0.8 0.7 3.7 5.4

3.2 2.2 7.1 12.7

0.8 0.9 4.2 5.4

32.5 38.1 33.5 49.7

26.6 35.4 26.5 67.1

ORF2a ORF2b ORF3 ORF4 ORF5 ORF6 ORF7 30 UTR

GP2 2b (E) GP3 GP4 GP5 M N

5.8 3.3 9.5 8.8 9.5 3.5 3.6 8.4

4.4 4.2 9.8 9.6 11.9 1.2 0.8

5.3 3.3 9.8 8.6 9.7 3.9 3.6 8.5

3.6 4.2 9.7 9.6 12.5 1.7 0.8

34.5 27.9 37.5 34.4 37.8 30.7 38.5 28.9

39.6 26.8 43.1 34.6 43.8 23.0 42.7

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Fig. 4. Phylogenetic relationship of a 2002 Korean field isolate (LMY) with field isolates of North American and European genotypes based on full genome sequence. Reliability of the tree was assessed by bootstrap analysis with 1000 replications.

amino acids. The Nsp9 and Nsp10, which are postulated to be RNA-dependent-RNA polymerase and helicase, respectively (Meulenberg et al., 1993; Wootton et al., 2000), were highly conserved whereas Nsp11 and Nsp12 showed relatively high sequence divergence from VR-2332. ORFs 2–7 contained 3188 nucleotides in length. In comparison to VR-2332, ORF5 of LMY showed the highest non-synonymous nucleotide changes from VR-2332 whereas ORF7 had the least variability. Detailed sequence divergence among LMY, VR-2332, PL97-1 and Lelystad for each ORF is summarized in Table 2. Phylogenetically, the full-length genomic sequence of the LMY strain showed the divergence of 5.7– 12.6% from type II PRRSVs (PL97-1, VR-2332, JA142, 16244B, P129, PA8, NVSL97-7895, CH-1a, HN1, BJ4, HB-1, and HB-2) but of 40.3–41.7% from type I viruses (Lelystad virus, U.S. EuroPRRSV). While PL97-1 was genetically classified to the VR2332 subgroup (16244B, BJ4, HN1, and PA8), the LMY strain was equally apart from the VR-2332 subgroup and China subgroup that was genetically closer to the JA142 strain (Fig. 4).

4. Discussion Comparative molecular epidemiology of a virus is valuable for developing a national control and prevention strategy for the virus and disease. In this study, all of the 2002–2003 Korean isolates examined were of the North American genotype. Based on the ORF5 sequence, all except four isolates were wild types even though virus isolation was attempted using MARC-145 cells which would favor isolating vaccine or vaccine-like viruses. As expected, a high degree of genetic variability was observed among the Korean PRRSVs and the degree of variability seemed to increase over time as demonstrated by other investigators (Chang et al., 2002; Cha et al., 2004). Since antigenic variations among PRRSVs due to genetic variability and its adverse impact on the protection by vaccination are well documented (Meng, 2000; Yang et al., 2000; Labarque et al., 2004), the observed genetic heterogeneity among the Korean PRRSVs and the fact that the majority of the virus isolates were of a wild type raises a concern about the efficacy of the MLV vaccine for PRRS control in Korea.

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The phylogenetic analyses of Korean, other Asian, North American and European PRRSVs revealed that the early and recent PRRSVs of the North American genotype in each of the Asian countries formed distinct clades along with geographical origin (Figs. 2 and 4). Although each clade showed closer relatedness to a different old prototype North American strain of PRRSV, PRRSVs reported from Asian countries including Korea were genetically distinct from more contemporary U.S. PRRSVs. These observations suggest that PRRSVs identified in different Asian countries likely have evolved independently from different parental strains, resulting in genetically distinct clades. Such a geographic differentiation among PRRSVs needs to be taken into consideration when control and preventive measures for international trade are established.

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