Expression Of EAV Minor Envelope Proteins Based On A PRRSV Infectious Clone

Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen causing serious economic losses in the global pig industry.In recent years,with its rapid evolution,more and more PRRSV strains have been found to have changed their adaptability to Marc-145 cells.In previous study,a chimeric clone pAPRRSV-EAV234 in which PRRSV GP2/GP3/GP4 coding region were swapped with EAV corresponding region was constructed.The chimeric virus not only can infect Marc-145 cells,but also could infect PRRSV non-susceptible cells BHK-21 and Vero cells.Furthermore,the chimeric virus lost the ability to infect PAM,which is susceptible to PRRSV.This study demonstrates that the viral protein that determines the PRRSV cell tropism of PRRSV cells is a GP2/GP3/GP4 complex or one of its proteins.In this study,related research on PRRSV and EAV GP2/GP3/GP4 were carried out to explore which protein determine the PRRSV cellular tropism.1.Construction and rescue of PRRSV recombinant clone expressing EAV GP2,GP3 or GP4Numerous studies have shown that GP2,GP3 or GP4 proteins of PRRSV are crucial to PRRSV infection.Using PRRSV infectious clone(pGXAM)as vector,the recombinant clones(pGXAM-E2,pGXAM-E3,pGXAM-E4)in which EAV GP2,GP3 or GP4 protein coding region was inserted between PRRSV ORF1 and ORF2 respectively.All recombinant clones can be rescued successfully on Marc-145 cells,recombinant viruses(vGXAM-E2,vGXAM-E3,vGXAM-E4)have the similar growth characteristics with the parent virus.The exogenous gene(EAV GP2,GP3 or GP4)is stable in the process of virus passage.However,all the recombinant viruses cannot infect BHK-21 cells.It is assumed that EAV GP2,GP3 or GP4 could not interact other PRRSV proteins to assembly into PRRSV virion.Based on the backbone of pGXAM-E2,pGXAM-E3 or pGXAM-E4,we then knocked out the translation initiation codon(ATG)of GP2,GP3 and GP4,respectively.The resulting recombinant clones pGXAM-2EN-E2,pGXAM-3KO-E3 and pGXAM-4KO-E4 could not be rescued on Marc-145 or BHK-21 cells.It is presumed that individual EAV minor envelope protein cannot be assembled into virus particles in a polymer with PRRSV proteins or individual envelope protein could not change the cell tropsim of PRRSV.2.Construction of Marc-145 cell lines expressing PRRSV or EAV GP2,GP3 or GP4The aim of this study was to use the lentiviral vector to construct Marc-145 cell lines expressing PRRSV or EAV GP2,GP3 and GP4 respectively.The coding regions of PRRSV or EAV GP2,GP3,GP4 were amplified by PCR from PRRSV and EAV infectious clone,then lentiviral vectors pLenti-PRRSV GP2/GP3/GP4-Hygro and pLenti-EAV GP2/GP3/GP4 were constructed by enzyme digestion and ligation.These recombinant lentiviral vectors were co-transfected with ps PAX2,VSVG into 293 T cell.At last,the attained packaged lentivirus was used to infect Marc-145 cell.PRRSV and EAV GP2/GP3/GP4 Marc-145 cell lines were screened by hygromycin B and endpoint dilution methods.The construction of these cell lines will lay a foundation for further research on basic structural proteins of arteritis virus,chimeric virus and cell tropism of arteritis virus.3.Construction and rescue of PRRSV nsp2 aa deletion infectious clonePreviously,we isolated a PRRSV strain with 155 aa deletion in nsp2.We then constructed a mutant(pGXAM-Nsp2-D155)with 155 aa deletion at the corresponding position of nsp2 based on a PRRSV infectious clone(pGXAM).The obtained mutant virus can be stably passaged and have a similar growth characteristics with parental virus vGXAM.We then constructed a mutant clone in which RFP gene was inserted at the deleted position.The resulting mutant clone(pGXAM-Nsp2-RFP)was rescued and passaged in Marc-145 cells.The mutant virus rGXAM-Nsp2-RFP expressed stably RFP in Marc-145.However biological assessments of recombinant virus,rGXAM-Nsp2-RFP showed that it maintained similar growth dynamics,yet yielded less infectious viruses when compared to the parental virus.This indicates that PRRSV nsp2 can tolerate 155 aa deletion and insertion of foreign genes.This study lays a foundation for development PRRSV marker vaccine.