Establishment of Cell Lines Persistently Infected with the Field High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

doi:10.3969/j.issn.1000-7091.2012.05.029

Abstract

Abstract: To establish an in vitro persistent infection cell model of high pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)based on Marc-145 cells.A Marc-145 cell line was infected with a field-isolated HP-PRRSV strain JX143 and then subcultured 35 passages.Hence a steady model of the Marc-145 cells persistently infected by PRRSV was established and the JX143-Marc-145-P35 cell line was obtained.Furthermore,RT-PCR,immunofluorescence assay and plaque assay were used to study the basic viral features of the persistently infected Marc-145 cells.The complete genes of JX143-Marc145-P35v was amplified and sequenced for further analysis the genetic changes involving the persistent infection process of PRRSV.During the process of persistent infection,we observed that the CPE occurs slower,then faster and slower in the end.Both RT-PCR and immunofluorescence assay showed positive results which indicated that PRRSV was quite stable in the JX143-Marc-145-P35 cell line and this cell line had the fundamental characteristics of persistently infected virus.There was no significant difference in their cell cycle between JX143-Marc-145-P35 cell line and the normal Marc-145,but in comparison with the parental virus,larger plaque size was observed.Comparative sequence analysis of the entire genomes of the JX143-Marc-145-P35v and JX143 identified 38 amino acid substitutions,including 31 in the replicase and 7 in the structural proteins.We conclude that persistent infection of the Mrac-145 cell line with PRRSV is established and that multiple genetic and phenotypic variations occur in the virus during persistence.The selective pressure for amino acid change is likely exerted in genes coding for the nonstructural protein and the major viral glycoproteins during establishment of persistence.This cell model can provide a valuable system for studying virus-cell interactions,especially the mechanism of persistent infection.

Key words: High pathogenic, Porcine reproductive and respiratory syndrome virus(HP-PRRSV), Persistent infection, Cell model

CLC Number:

S858.28

WANG Xiao-min, LI Yan-hua, LIN Tao, LIU Run-xia, HE Kong-wang, TONG Guang-zhi, YUAN Shi-shan. Establishment of Cell Lines Persistently Infected with the Field High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(5): 150-156. doi: 10.3969/j.issn.1000-7091.2012.05.029.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.3969/j.issn.1000-7091.2012.05.029

http://www.hbnxb.net/EN/Y2012/V27/I5/150

References

[1] Rossow K D. Porcine Reproductive and Respiratory Syndrome[J]. Veterinary Pathology, 1998,35 : 1 20.

[2] Meulenberg J J, Petersen den Besten A, De Kluyver E, et al. Molecular characterization of Lelystad virus[Jl. Veterinary Microbiology, 1997,55 : 197 202.

[3] Hall W. Porcine reproductive and respiratory syndrome (PRRS) virus-a significant disease of pigs[J]. Australian Veterinary Journal ,2005,83 ( 5 ) :260 261.

[4] Keffaber K K. Reproductive failure of unknown etiology [J] American Association of Swine Practitioners (News),1989 1:1 -9.＜br＞
[5] Wensvoort G, Terpstra C, Pol J M, et al. Mystery swine disease in the Netherlands:the isolation of Lelystad virus [J]. Veterinary Quarterly, 1991,13 ( 3 ) : 121 130.

[6] Tian K G, Yu X L,Zhao T Z,et al. Emergence of fatal PRRSV variants : unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark [J]. PloS ONE,2007,2(6) :526 -539.

[7] Tong G Z,Zhou Y J, Hao X F,et al. Highly pathogenic porcine reproductive and respiratory syndrome, China [J]. Emerging Infeeioust Diseases ,2007,13 ( 9 ) : 1434 1436.

[8] Suarez P. Ultrastructural pathogenesis of the PRRS virus [J]. Veterinary Research,2000,31 ( 1 ) :47 -55.

[9] Allende R, Laegreid W W,Kutish G F,et al. Porcine re-productive and respiratory syndrome virus:description of persistence in individual pigs upon experimental infection[J]. Journal of Virology, 2000,74 ( 22 ) : 10834 10837.

[10] Christopher-Hennings J, et al. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars [J]. Journal of Veterinary Diagnostic Investigation, 1995,7:456 464.

[11] Costers S, Delputte P L, Nauwynck H J. Porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis [J]. Journal of General Virology, 2006, 87:2341 2351.

[12] Tjeerd G K, Lisette A C, Rob J M,et al. Challenges for porcine reproductive and respiratory syndrome virus ( PRRSV ) vaeeinology [J]. Vaccine, 2009 ( 27 ) : 3704 3718.

[13] Baybutt H N,Wege H,Carter M J,et al. Adaptation of coronavirus JHM to persistent injection of routine sac ( ) cells [J]. Journal of General Virology, 1984,65 (5) :915 -24.

[14] Lamontagne L M, Dupuy J M. Persistent infection with mouse hepatitis virus 3 in mouse lynlphoid cell lines [J]. Infection and immunity,1984,44(3):716 -723.

[15] Chan P K,To K F,Lo A W,et al. Persistent infection of SARS coronavirus in colonic cells in vitro[J]. Journal of Medical Virology ,2004,74( 1 ) : 1 7.

[16] Yamate M, Yamashita M, Goto T, et al. Establishment of vero E6 cell clones persistently infected with severe acute respiratory syndrome corenavirus [J]. Microbes and Infection ,2005,7 ( 15 ) : 1530 1540.

[17] Zhang J Q, Timoney P J, James MaeLachlan N, et al. Persistent equine arteritis virus infection in Hela cells [J]. Journal of Virology ,2008,82( 17 ) :8456 8464.

[18] Colb re-Garapin F, Christodoulou C, Crainie R, et al.Persistent poliovirus infection of human neuroblastoma cells[J]. PNAS,1989,86(19) :7590 -7594.

[19] Torre J C,Martinez-Salas E, Diez J,et al. Extensive cell heterogeneity during persistent infection with foot-and mouth disease virus [J]. Journal of Virology, 1989,63 (1) :59 -63.

[20] Duncan G, Pelletier I, Colb re-Garapin F. Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c ceils by modifying virus-receptor interactions [J]. Virology, 1998,241 : 14 29.

[21] Hedges J F,Balasuriya U B,Timoney P J,et al. Genetic divergence with emergence of novel phenotypic variants of equine arteritis virus during persistent infection of stallions [J]. Journal of Virology, 1999,73 : 3672 3681.

[22] Brown E, Lawson S, Welbon C, et al. Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types I and II[J]. Clinical and vaccine immunology, 2009, 16 (5) :628 -635.

[23] Murtaugh M P, Elam M R, Kakach L T. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus [J]. Archives of Virology, 1995,140 : 1451 1460.

[24] Gall L, Albina E, Magar R, et al. Antigenic variability of porcine reproductive and respiratory syndrome (PRRS) virus isolates. Influence of virus passage in pig[J]. Veterinary Researeh, 1997,28 ( 3 ) : 247 257.

[25] Rowland R R, Steffen M, Ackerman T, et al. The evolution of porcine reproductive and respiratory syndrome virus : quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332 [J]. Virology, 1999,259:262 266.

Please choose a citation manager

Content to export