Abstract
In recent years, the availability of reverse genetics systems for porcine reproductive and respiratory syndrome virus (PRRSV) has created new perspectives for the use of recombinant viruses as expression vectors. Most of these recombinant PRRSV vectors express foreign genes through either an independent transcription unit inserted in ORF1b and ORF2, or in ORF7 and the 3′ UTR. The aim of this study was to find an alternative site for foreign gene insertion into the PRRSV genome. Here, we constructed an infectious cDNA clone for a cell-adapted PRRSV strain, GXNN1396-P96. This cDNA-clone-derived recombinant virus (rGXAM) was comparable in its growth kinetics in MARC-145 cells to the parental virus, GX1396-P96. Using the infectious cDNA-clone, we inserted an independent transcription unit in ORF4 and ORF5a to generate a novel PRRSV-based recombinant virus expressing the green fluorescent protein (GFP) gene. Biological characterization of the recombinant virus, rGX45BSTRS-GFP, showed that it maintained similar growth characteristics but produced fewer infectious virions than the parental PRRSV. These data demonstrate that the ORF4 and ORF5a site is able to tolerate the insertion of foreign genes.
Similar content being viewed by others
References
Cao QM, Ni YY, Cao D, Tian D, Yugo DM, Heffron CL, Overend C, Subramaniam S, Rogers AJ, Catanzaro N, LeRoith T, Roberts PC, Meng XJ (2018) Recombinant porcine reproductive and respiratory syndrome virus expressing membrane-bound interleukin-15 as an immunomodulatory adjuvant enhances NK and gammadelta T cell responses and confers heterologous protection. J Virol 92
Chen Z, Zhou X, Lunney JK, Lawson S, Sun Z, Brown E, Christopher-Hennings J, Knudsen D, Nelson E, Fang Y (2010) Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response. J Gen Virol 91:1047–1057
Choi HW, Nam E, Lee YJ, Noh YH, Lee SC, Yoon IJ, Kim HS, Kang SY, Choi YK, Lee C (2014) Genomic analysis and pathogenic characteristics of Type 2 porcine reproductive and respiratory syndrome virus nsp2 deletion strains isolated in Korea. Vet Microbiol 170:232–245
de Vries AA, Glaser AL, Raamsman MJ, de Haan CA, Sarnataro S, Godeke GJ, Rottier PJ (2000) Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope. Virology 270:84–97
de Vries AA, Glaser AL, Raamsman MJ, Rottier PJ (2001) Recombinant equine arteritis virus as an expression vector. Virology 284:259–276
Di H, Morantz EK, Sadhwani H, Madden JC Jr, Brinton MA (2018) Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV). Virology 525:150–160
Fang Y, Rowland RR, Roof M, Lunney JK, Christopher-Hennings J, Nelson EA (2006) A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region. J Virol 80:11447–11455
Fang Y, Christopher-Hennings J, Brown E, Liu H, Chen Z, Lawson SR, Breen R, Clement T, Gao X, Bao J, Knudsen D, Daly R, Nelson E (2008) Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development. J Gen Virol 89:3086–3096
Fang Y, Snijder EJ (2010) The PRRSV replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins. Virus Res 154:61–76
Firth AE, Zevenhoven-Dobbe JC, Wills NM, Go YY, Balasuriya UBR, Atkins JF, Snijder EJ, Posthuma CC (2011) Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production. J Gen Virol 92:1097–1106
Gao F, Qu Z, Li L, Yu L, Jiang Y, Zhou Y, Yang S, Zheng H, Huang Q, Tong W, Tong G (2016) Recombinant porcine reproductive and respiratory syndrome virus expressing luciferase genes provide a new indication of viral propagation in both permissive and target cells. Res Vet Sci 107:132–140
Gao F, Jiang Y, Li G, Zhou Y, Yu L, Li L, Tong W, Zheng H, Zhang Y, Yu H, Shan T, Yang S, Liu H, Zhao K, Tong G (2018) Porcine reproductive and respiratory syndrome virus expressing E2 of classical swine fever virus protects pigs from a lethal challenge of highly-pathogenic PRRSV and CSFV. Vaccine 36:3269–3277
Han J, Wang Y, Faaberg KS (2006) Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus. Virus Res 122:175–182
Han J, Liu G, Wang Y, Faaberg KS (2007) 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 81:9878–9890
Han M, Ke H, Du Y, Zhang Q, Yoo D (2017) Reverse genetics for porcine reproductive and respiratory syndrome virus. Methods Mol Biol 1602:29–46
He W, Wei Y, Yao J, Xie X, Huang J, Lin S, Ouyang K, Chen Y, Huang W, Wei Z (2018) Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro. Arch Virol 163:1489–1501
Hong S, Wei Y, Lin S, Huang J, He W, Yao J, Chen Y, Kang O, Huang W, Wei Z (2019) Genetic analysis of porcine productive and respiratory syndrome virus between 2013 and 2014 in Southern parts of China: identification of several novel strains with amino acid deletions or insertions in nsp2. BMC Vet Res 15:171
Johnson CR, Griggs TF, Gnanandarajah J, Murtaugh MP (2011) Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses. J Gen Virol 92:1107–1116
Kedkovid R, Nuntawan Na Ayudhya S, Amonsin A, Thanawongnuwech R (2010) NSP2 gene variation of the North American genotype of the Thai PRRSV in central Thailand. Virol J 7:340
Kim DY, Kaiser TJ, Horlen K, Keith ML, Taylor LP, Jolie R, Calvert JG, Rowland RR (2009) Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity. Virus Genes 38:118–128
Lee C, Calvert JG, Welch SK, Yoo D (2005) A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology 331:47–62
Li Z, Wang G, Wang Y, Zhang C, Huang B, Li Q, Li L, Xue B, Ding P, Cai X, Wang C, Zhou EM (2015) Immune responses of pigs immunized with a recombinant porcine reproductive and respiratory syndrome virus expressing porcine GM-CSF. Vet Immunol Immunopathol 168:40–48
Li Z, Wang G, Wang Y, Zhang C, Wang X, Huang B, Li Q, Li L, Xue B, Ding P, Syed SF, Wang C, Cai X, Zhou EM (2015) Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4. Virol J 12:185
Lv J, Zhang J, Sun Z, Liu W, Yuan S (2008) An infectious cDNA clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome. J Gen Virol 89:2075–2079
Meulenberg JJ, Bos-de Ruijter JN, Wensvoort G, Moormann RJ (1998) An infectious cDNA clone of porcine reproductive and respiratory syndrome virus. Adv Exp Med Biol 440:199–206
Nelsen CJ, Murtaugh MP, Faaberg KS (1999) Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J Virol 73:270–280
Neumann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ (2005) Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 227:385–392
Pasternak AO, Spaan WJ, Snijder EJ (2006) Nidovirus transcription: how to make sense…? J Gen Virol 87:1403–1421
Pei Y, Hodgins DC, Wu J, Welch SK, Calvert JG, Li G, Du Y, Song C, Yoo D (2009) Porcine reproductive and respiratory syndrome virus as a vector: immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs. Virology 389:91–99
Sang Y, Shi J, Sang W, Rowland RR, Blecha F (2012) Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) viruses expressing indicator proteins and antiviral cytokines. Viruses 4:102–116
Snijder EJ, Meulenberg JJ (1998) The molecular biology of arteriviruses. J Gen Virol 79(Pt 5):961–979
Sun L, Li Y, Liu R, Wang X, Gao F, Lin T, Huang T, Yao H, Tong G, Fan H, Wei Z, Yuan S (2013) Porcine reproductive and respiratory syndrome virus ORF5a protein is essential for virus viability. Virus Res 171:178–185
Tian D, Zheng H, Zhang R, Zhuang J, Yuan S (2011) Chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2. Virology 412:1–8
Tian D, Sooryanarain H, Matzinger SR, Gauger PC, Karuppannan AK, Elankumaran S, Opriessnig T, Meng XJ (2017) Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus. J Gen Virol 98:3026–3036
Truong HM, Lu Z, Kutish GF, Galeota J, Osorio FA, Pattnaik AK (2004) A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus. Virology 325:308–319
van Marle G, Dobbe JC, Gultyaev AP, Luytjes W, Spaan WJ, Snijder EJ (1999) Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proc Natl Acad Sci USA 96:12056–12061
Wang C, Huang B, Kong N, Li Q, Ma Y, Li Z, Gao J, Zhang C, Wang X, Liang C, Dang L, Xiao S, Mu Y, Zhao Q, Sun Y, Almazan F, Enjuanes L, Zhou EM (2013) A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit. Vet Res 44:104
Wang C, Meng H, Gao Y, Gao H, Guo K, Almazan F, Sola I, Enjuanes L, Zhang Y, Abrahamyan L (2017) Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Vet Res 48:41
Wang L, Hou J, Gao L, Guo XK, Yu Z, Zhu Y, Liu Y, Tang J, Zhang H, Feng WH (2014) Attenuation of highly pathogenic porcine reproductive and respiratory syndrome virus by inserting an additional transcription unit. Vaccine 32:5740–5748
Wang X, Sun L, Li Y, Lin T, Gao F, Yao H, He K, Tong G, Wei Z, Yuan S (2013) 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 171:150–160
Wang Y, Liang Y, Han J, Burkhart KM, Vaughn EM, Roof MB, Faaberg KS (2008) Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence. Virology 371:418–429
Wei Z, Tian D, Sun L, Lin T, Gao F, Liu R, Tong G, Yuan S (2012) Influence of N-linked glycosylation of minor proteins of porcine reproductive and respiratory syndrome virus on infectious virus recovery and receptor interaction. Virology 429:1–11
Wissink EH, Kroese MV, van Wijk HA, Rijsewijk FA, Meulenberg JJ, Rottier PJ (2005) Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus. J Virol 79:12495–12506
Wu WH, Fang Y, Farwell R, Steffen-Bien M, Rowland RR, Christopher-Hennings J, Nelson EA (2001) A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b. Virology 287:183–191
Yoshii M, Okinaga T, Miyazaki A, Kato K, Ikeda H, Tsunemitsu H (2008) Genetic polymorphism of the nsp2 gene in North American type–porcine reproductive and respiratory syndrome virus. Arch Virol 153:1323–1334
Yu D, Lv J, Sun Z, Zheng H, Lu J, Yuan S (2009) Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus. Virology 383:22–31
Yuan S, Wei Z (2008) Construction of infectious cDNA clones of PRRSV: separation of coding regions for nonstructural and structural proteins. Sci China C Life Sci 51:271–279
Zheng H, Sun Z, Zhu XQ, Long J, Lu J, Lv J, Yuan S (2010) Recombinant PRRSV expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus. Virus Res 148:8–16
Zheng H, Zhang K, Zhu XQ, Liu C, Lu J, Gao F, Zhou Y, Zheng H, Lin T, Li L, Tong G, Wei Z, Yuan S (2014) Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity. Arch Virol 159:1927–1940
Zhou L, Zhang J, Zeng J, Yin S, Li Y, Zheng L, Guo X, Ge X, Yang H (2009) The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence. J Virol 83:5156–5167
Acknowledgements
The authors would like to thank Dr. Dev Sooranna, Imperial College, London, for editing the manuscript.
Funding
This work was funded by the National Natural Science Foundation of China (nos. 31660716 and 31372444) and the Guangxi Natural Science Foundation (no. 2018GXNSFDA281021).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare they have no conflict of interest.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Handling Editor: Sheela Ramamoorthy.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Wang, Y., He, W., Li, Q. et al. Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a. Arch Virol 165, 1803–1813 (2020). https://doi.org/10.1007/s00705-020-04679-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00705-020-04679-3