J Vet Sci.  2018 May;19(3):358-367. 10.4142/jvs.2018.19.3.358.

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages

• Lee SC
• Lee S
• Yoo GW
• Choi HW
• Noh YH
• Park CE
• Shin JH
• Yoon IJ
• Kang SY
• Lee C

• KMID: 2412126
• DOI: http://doi.org/10.4142/jvs.2018.19.3.358

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

Keyword

attenuated vaccines; macrophage tropism; porcine reproductive and respiratory syndrome virus; virulence; whole genome sequencing

MeSH Terms

Animals
Antibody Formation
Body Temperature
Genetic Drift
Humans
Kinetics
Macrophages
Macrophages, Alveolar*
Parents
Phenotype
Porcine Reproductive and Respiratory Syndrome*
Porcine respiratory and reproductive syndrome virus*
Swine
Tropism
Vaccines, Attenuated
Viremia
Virulence
Weight Gain
Vaccines, Attenuated

Figure

• 

  Fig. 1 Average body temperature (A) and average daily weight gain (B) of pigs after challenge with the CA-2-P20, CA-2-P100, or CA-2-MP120 strains of porcine reproductive and respiratory syndrome virus or mock challenge with culture medium. Rectal temperatures equal to or greater than 40℃ were defined as fever. Error bars represent SD. “a” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-P100 groups; “b” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-MP120 groups.

• 

  Fig. 2 Virus replication in vivo and seroconversion. (A) Mean levels of viremia of pigs infected with CA-2-P20, CA-2-P100, or CA-2-MP120. TCID50, 50% tissue culture infectious dose. (B) Virus-specific antibody response of pigs measured by using a commercial enzyme-linked immunosorbent assay kit. Serum samples were considered positive for antibodies to porcine reproductive and respiratory syndrome virus if the sample:positive (S/P) ratio was equal to or greater than 0.4. Error bars represent SD. “a” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-P100 groups; “b” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-MP120 groups; “c” denotes a significant difference (p < 0.05) between the CA-2-P100 and CA-2-MP120 groups.

• 

  Fig. 3 Photomicrographs of the lungs and lymph nodes (LN) from pigs in the CA-2-P20, CA-2-P100, CA-2-MP120, and control groups. H&E-stained tissues from the pigs in each group were histopathologically examined. 100×. The upper panels represent lung sections from pigs of each group. A CA-2-P20-infected pig (Group 1) shows a marked interstitial pneumonia with alveolar walls thickened by massive lymphocyte and macrophage infiltration surrounding perivascular cuffing (arrows), whereas pigs inoculated with CA-2-P100 (Group 2) or CA-2-MP120 (Group 3) exhibit normal lung lesions comparable to control pig lung. The lower panels represent LN sections from pigs of each group. A pig in Group 1 displayed lymphoid follicular hyperplasia (arrow), whereas pigs in Groups 2 and 3 exhibited no remarkable LN lesions, as was observed in control pig LN samples.

• 

  Fig. 4 Average histopathologic scores of lung (A) and lymph node (B) tissues from pigs in the CA-2-P20, CA-2-P100, CA-2-MP120, and control groups. The results are expressed as mean values from 6 pigs in each group, and error bars represent SD. *p = 0.001–0.05, **p < 0.001.

• 

  Fig. 5 Schematic representation of amino acid (aa) differences between the parental CA-2-P20 virus and its cell-adapted derivatives. The genomic sequences of the virus strains were determined as described in Table 1. The organization of the CA-2 genome (approximately 15 kb) is shown at the top. The coding regions for each nonstructural protein (nsp) in open reading frames (ORFs) 1a and 1b and for each structural protein are indicated. The lower panels show the genomes of the parental CA-2-P20 strain and its cell-adapted derivatives, where the vertical lines (black) represent a 1 aa substitution relative to the predicted aa sequence of CA-2-P20. The vertical dotted lines denote a 1 aa substitution in the overlapping coding regions for nsp2/nsp2TF, GP2/E, and GP5/ORF5a (silent mutation in nsp2, whereas a non-silent mutation in nsp2TF; silent mutation in GP2, whereas a non-silent mutation in E; non-silent mutation in GP5, whereas a silent mutation in ORF5a). The vertical lines (red) in the PAM-passaged CA-2-MP-120 strain indicate 15 aa changes relative to the predicted aa sequence of CA-2-P100 and the vertical arrows (red) indicate reverting mutations that restore the sequence of CA-2-P20.

Reference

1. Adams MJ, Lefkowitz EJ, King AMQ, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR, Nibert M, Sabanadzovic S, Sanfaçon H, Siddell SG, Simmonds P, Varsani A, Zerbini FM, Gorbalenya AE, Davison AJ. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch Virol. 2017; 162:2505–2538.

2. Allende R, Kutish GF, Laegreid W, Lu Z, Lewis TL, Rock DL, Friesen J, Galeota JA, Doster AR, Osorio FA. Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype. Arch Virol. 2000; 145:1149–1161.

3. Beura LK, Sarkar SN, Kwon B, Subramaniam S, Jones C, Pattnaik AK, Osorio FA. Porcine reproductive and respiratory syndrome virus nonstructural protein 1β modulates host innate immune response by antagonizing IRF3 activation. J Virol. 2010; 84:1574–1584.

4. Brockmeier SL, Loving CL, Vorwald AC, Kehrli ME Jr, Baker RB, Nicholson TL, Lager KM, Miller LC, Faaberg KS. Genomic sequence and virulence comparison of four type 2 porcine reproductive and respiratory syndrome virus strains. Virus Res. 2012; 169:212–221.

5. Chang CC, Yoon KJ, Zimmerman JJ, Harmon KM, Dixon PM, Dvorak CM, Murtaugh MP. Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs. J Virol. 2002; 76:4750–4763.

6. Choi HW, Nam E, Lee YJ, Noh YH, Lee SC, Yoon IJ, Kim HS, Kang SY, Choi YK, Lee C. Genomic analysis and pathogenic characteristics of type 2 porcine reproductive and respiratory syndrome virus nsp2 deletion strains isolated in Korea. Vet Microbiol. 2014; 170:232–245.

7. Das PB, Dinh PX, Ansari IH, de Lima M, Osorio FA, Pattnaik AK. The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD163. J Virol. 2010; 84:1731–1740.

8. Faaberg KS, Han J, Wang Y. Molecular dissection of porcine reproductive and respiratory virus putative nonstructural protein 2. Adv Exp Med Biol. 2006; 581:73–77.

9. Fang Y, Kim DY, Ropp S, Steen P, Christopher-Hennings J, Nelson EA, Rowland RR. Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States. Virus Res. 2004; 100:229–235.

10. Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, Lum MA, Andrews JJ, Rathje JA. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1995; 32:648–660.

11. Han J, Liu G, Wang Y, Faaberg KS. Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture. J Virol. 2007; 81:9878–9890.

12. Han J, Wang Y, Faaberg KS. Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus. Virus Res. 2006; 122:175–182.

13. Holtkamp DJ, Kliebenstein JB, Neumann EJ, Zimmerman JJ, Rotto HF, Yoder TK, Wang C, Yeske PE, Mowrer CL, Haley CA. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. J Swine Health Prod. 2013; 21:72–84.

14. Kappes MA, Faaberg KS. PRRSV structure, replication and recombination: origin of phenotype and genotype diversity. Virology. 2015; 479-480:475–486.

15. Keffaber KK. Reproductive failure of unknown etiology. Am Assoc Swine Pract Newsl. 1989; 1:1–9.

16. Kim HK, Lee CS, Kang BK, Yeom MJ, Moon HJ, Park SJ, Nguyen VG, Song DS, Park BK. Experimental infection of a newly emerging Korean type I porcine reproductive and respiratory syndrome virus isolate in colostrum-deprived pigs. Virol J. 2011; 8:177.

17. Kim JY, Lee SY, Sur JH, Lyoo YS. Serological and genetic characterization of the European strain of the porcine reproductive and respiratory syndrome virus isolated in Korea. Korean J Vet Res. 2006; 46:363–370.

18. Kim SH, Roh IS, Choi EJ, Lee C, Lee CH, Lee KH, Lee KK, Song YK, Lee OS, Park CK. A molecular analysis of European porcine reproductive and respiratory syndrome virus isolated in South Korea. Vet Microbiol. 2010; 143:394–400.

19. Kweon CH, Kwon BJ, Lee HJ, Cho JJ, Hwang EK, Shin JH, Yoon YD, Kang YB, An SH, Kim YH, Huh W, Jun MH, Wensvoort G. Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in Korea. Korean J Vet Res. 1994; 34:77–83.

20. Kwon B, Ansari IH, Pattnaik AK, Osorio FA. Identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones. Virology. 2008; 380:371–378.

21. Lee S, Lee C. Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerg Infect Dis. 2014; 20:1223–1226.

22. Lee SC, Choi HW, Nam E, Noh YH, Lee S, Lee YJ, Park GS, Shin JH, Yoon IJ, Kang SY, Lee C. Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2. Vet Microbiol. 2016; 186:174–188.

23. Lee YJ, Park CK, Nam E, Kim SH, Lee OS, Lee DS, Lee C. Generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus. J Virol Methods. 2010; 163:410–415.

24. Martínez-Lobo FJ, Díez-Fuertes F, Segalés J, García-Artiga C, Simarro I, Castro JM, Prieto C. Comparative pathogenicity of type 1 and type 2 isolates of porcine reproductive and respiratory syndrome virus (PRRSV) in a young pig infection model. Vet Microbiol. 2011; 154:58–68.

25. Nam E, Park CK, Kim SH, Joo YS, Yeo SG, Lee C. Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea. Arch Virol. 2009; 154:629–638.

26. Nelsen CJ, Murtaugh MP, Faaberg KS. Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J Virol. 1999; 73:270–280.

27. Sagong M, Park CK, Kim SH, Lee KK, Lee OS, Lee DS, Cha SY, Lee C. Human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus. J Virol Methods. 2012; 179:26–32.

28. Shi M, Lam TT, Hon CC, Murtaugh MP, Davies PR, Hui RK, Li J, Wong LT, Yip CW, Jiang JW, Leung FC. Phylogeny-based evolutionary demographical and geographical dissection of North American type 2 porcine reproductive and respiratory syndrome viruses. J Virol. 2010; 84:8700–8711.

29. Snijder EJ, Kikkert M, Fang Y. Arterivirus molecular biology and pathogenesis. J Gen Virol. 2013; 94:2141–2163.

30. Sun Y, Han M, Kim C, Calvert JG, Yoo D. Interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus. Viruses. 2012; 4:424–446.

31. Wang X, Sun L, Li Y, Lin T, Gao F, Yao H, He K, Tong G, Wei Z, Yuan S. Development of a differentiable virus via a spontaneous deletion in the nsp2 region associated with cell adaptation of porcine reproductive and respiratory syndrome virus. Virus Res. 2013; 171:150–160.

32. Wissink EH, Kroese MV, van Wijk HA, Rijsewijk FA, Meulenberg JJ, Rottier PJ. Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus. J Virol. 2005; 79:12495–12506.

33. Yuan S, Mickelson D, Murtaugh MP, Faaberg KS. Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains. Virus Res. 2001; 79:189–200.

34. Zimmerman JJ, Benfield DA, Dee SA, Murtaugh MP, Stadejek T, Stevenson GW, Torremorell M. Porcine reproductive and respiratory syndrome virus (porcine arterivirus). In : Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, editors. Diseases of Swine. 10th ed. Ames: Wiley-Blackwell;2012. p. 461–486.