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
Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. The limitations of current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of type 1 PRRSV, this study constructed a recombinant green fluorescent protein (GFP)-tagged PRRSV containing a deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viraemia in comparison with that of the parental virus. To complement the marker identification, GFP and ES4 epitope-based ELISAs were developed. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, but generated a high-level antibody response to GFP by 14 days post-infection. These results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRS.
AllendeR.,
LewisT. L.,
LuZ.,
RockD. L.,
KutishG. F.,
AliA.,
DosterA. R.,
OsorioF. A.1999; North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J Gen Virol 80:307–315
BautistaE. M.,
GoyalS. M.,
SoonI. J.,
JooH. S.,
CollinsJ. E.1996; Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus. Arch Virol 141:1357–1365[CrossRef]
BoschJ. C.,
KaashoekM. J.,
KroeseA. H.,
Van OirschotJ. T.1996; An attenuated bovine herpesvirus 1 marker vaccine induces a better protection than two inactivated marker vaccines. Vet Microbiol 52:223–234[CrossRef]
Castillo-OlivaresJ.,
WieringaR.,
BakonyiT.,
de VriesA. A.,
Davis-PoynterN. J.,
RottierP. J.2003; Generation of a candidate live marker vaccine for equine arteritis virus by deletion of the major virus neutralization domain. J Virol 77:8470–8480[CrossRef]
de LimaM.,
PattnaikA. K.,
FloresE. F.,
OsorioF. A.2006; Mapping of B-cell linear epitopes on Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology 353:410–421[CrossRef]
de LimaM.,
KwonB.,
AnsariI. H.,
PattnaikA. K.,
FloresE. F.,
OsorioF. A.2008; Development of a porcine reproductive and respiratory syndrome virus differentiable (DIVA) strain through deletion of specific immunodominant epitopes. Vaccine 26:3594–3600[CrossRef]
den BoonJ. A.,
FaabergK. S.,
MeulenbergJ. J. M.,
WassenaarA. L. M.,
PlagemannP. G. W.,
GorbelenyaA. E.,
SnijderE. J.1995; Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papainlike cysteine proteases. J Virol 69:4500–4505
FangY.,
KimD. Y.,
RoppS.,
SteenP.,
Christopher-HenningsJ.,
NelsonE. A.,
RowlandR. R. R.2004; Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States. Virus Res 100:229–235[CrossRef]
FangY.,
RowlandR. R. R.,
RoofM.,
LunneyJ. K.,
Christopher-HenningsJ.,
NelsonE. A.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[CrossRef]
FangY.,
SchneiderP.,
ZhangW. P.,
FaabergK.,
NelsonE. A.,
RowlandR. R. R.2007; Diversity and evolution of a newly emerged North American Type 1 porcine arterivirus. Arch Virol 152:1009–1017[CrossRef]
FerrinN. H.,
FangY.,
JohnsonC. R.,
MurtaughM. P.,
PolsonD. D.,
TorremorellM.,
GramerM. L.,
NelsonE. A.2004; Validation of a blocking ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol 11:503–514
GaoZ. Q.,
GuoX.,
YangH. C.2004; Genomic characterization of two Chinese isolates of Porcine respiratory and reproductive syndrome virus . Arch Virol 149:1341–1351
HanJ.,
LiuG.,
WangY.,
FaabergK. S.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[CrossRef]
HayashiN.,
WelschofM.,
ZeweM.,
BraunagelM.,
DübelS.,
BreitlingF.,
LittleM.1994; Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. Biotechniques 17:310–315
KaashoekM. J.,
MoermanA.,
MadicJ.,
RijsewijkF. A.,
QuakJ.,
GielkensA. L.,
van OirschotJ. T.1994; A conventionally attenuated glycoprotein E-negative strain of bovine herpesvirus type 1 is an efficacious and safe vaccine. Vaccine 12:439–444[CrossRef]
KimD. Y.,
CalvertJ. G.,
ChangK. O.,
HorlenK.,
KerriganM.,
RowlandR. R. R.2007; Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV). Virus Res 128:106–114[CrossRef]
LabarqueG.,
Van ReethK.,
NauwynckH.,
DrexlerC.,
Van GuchtS.,
PensaertM.2004; Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy. Vaccine 22:4183–4190[CrossRef]
LawsonS.,
FangY.,
RowlandR. R. R.,
Christopher-HenningsJ.,
NelsonE. A.2005; Experimental infection of pigs with European-like (Type 1) PRRSV isolates of US origin. In The 86th Annual Meeting of the Conference of Research Workers in Animal Diseases abstract no: 994–6December 2005 St Louis; Missouri, USA:
MardassiH.,
MassiveB.,
DeaS.1996; Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus. Virology 221:98–112[CrossRef]
MartelliP.,
CordioliP.,
AlboraliL. G.,
GozioS.,
De AngelisE.,
FerrariL.,
LombardiG.,
BorghettiP.2007; Protection and immune response in pigs intradermally vaccinated against porcine reproductive and respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) field strain. Vaccine 25:3400–3408[CrossRef]
MebatsionT.,
KoolenM. J. M.,
de VaanL. T. C.,
de HaasN.,
BraberM.,
Romer-OberdorferA.,
van den ElzenP.,
van der MarelP.2002; Newcastle disease virus marker vaccine: an immunodominant epitope on the nucleoprotein gene of NDV can be deleted or replaced by a foreign epitope. J Virol 76:10138–10146[CrossRef]
MengX. J.,
PaulP. S.,
MorozovI.,
HalburP. G.1996; A nested set of six or seven subgenomic mRNAs is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus. J Gen Virol 77:1265–1270[CrossRef]
MengelingW. L.,
LagerK. M.,
VorwaldA. C.1999; Safety and efficacy of vaccination of pregnant gilts against porcine reproductive and respiratory syndrome. Am J Vet Res 60:796–801
MeulenbergJ. J.,
Petersen-den BestenA.1996; Identification and characterization of a sixth structural protein of Lelystad virus: the glycoprotein GP2 encoded by ORF2 is incorporated in virus particles. Virology 225:44–51[CrossRef]
MeulenbergJ. J.,
HulstM. M.,
de MeijerE. J.,
MoonenP. L.,
den BestenA.,
de KluyverE. P.,
WensvoortG.,
MoormannR. J.1993; Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192:62–72[CrossRef]
MeulenbergJ. J.,
Petersen-den BestenA.,
de KluyverA.,
MoormannE. P.,
SchaaperR. J. M.,
WensvoortG.1995; Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206:155–163[CrossRef]
MoormannR. J.,
de RoverT.,
BriaireJ.,
PeetersB. P.,
GielkensA. L.,
van OirschotJ. T.1990; Inactivation of the thymidine kinase gene of a g1 deletion mutant of pseudorabies virus generates a safe but still highly immunogenic vaccine strain. J Gen Virol 71:1591–1595[CrossRef]
MounirS.,
MardassiH.,
DeaS.1995; Identification and characterization of the porcine reproductive and respiratory virus ORFs 7, 5 and 4 products. Adv Exp Med Biol 380:317–320
NelsonE. A.,
Christopher-HenningsJ.,
WensvoortG.,
CollinsJ. E.,
BenfieldD. A.1993; Differentiation of United States and European isolates of porcine reproductive and respiratory syndrome (PRRS) virus using monoclonal antibodies. J Clin Microbiol 31:3184–3189
NeumannE. J.,
KliebensteinJ. B.,
JohnsonC. D.,
MabryJ. W.,
BushE. J.,
SeitzingerA. H.,
GreenA. L.,
ZimmermanJ. J.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[CrossRef]
OleksiewiczM. B.,
BotnerA.,
ToftP.,
NormannP.,
StorgaardT.2001; Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes. J Virol 75:3277–3290[CrossRef]
OsorioF. A.,
ZuckermannF.,
WillsR.,
MeierW.,
ChristianS.,
GaleotaJ.,
DosterA.1998; PRRSV: comparison of commercial vaccines in their ability to induce protection against current PRRSV strains of high virulence. Allen D Leman Swine Conf 25:176–182
RanZ. G.,
ChenX. Y.,
GuoX.,
GeX. N.,
YoonK. J.,
YangH. C.2008; Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region. Arch Virol 153:899–907[CrossRef]
RoppS. L.,
Mahlum WeesC. E.,
FangY.,
NelsonE. A.,
RossowK. D.,
BienM.,
ArndtB.,
PreszlerS.,
SteenP.other authors2004; Characterization of emerging European-like PRRSV isolates in the United States. J Virol 78:3684–3703[CrossRef]
ShenS.,
KwangJ.,
LiuW.,
LiuD. X.2000; Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion. Arch Virol 145:871–883[CrossRef]
SnijderE. J.,
WassenaarA. L. M.,
SpaanW. J. M.1994; Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J Virol 68:5755–5764
SnijderE. J.,
WassenaarA. L. M.,
SpaanW. J.,
GorbalenyaA. E.1995; The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases. J Biol Chem 270:16671–16676[CrossRef]
SunT.,
LuP.,
WangX.2004; Localization of infection-related epitopes on the non-structural protein 3ABC of foot-and-mouth disease virus and the application of tandem epitopes. J Virol Methods 119:79–86[CrossRef]
TianK.,
YuX.,
ZhaoT.,
FengY.,
CaoZ.,
WangC.,
HuY.,
ChenX.,
HuD.other authors2007; Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE 2:e526[CrossRef]
van GennipH. G.,
BoumaA.,
van RijnP. A.,
WidjojoatmodjoM. N.,
MoormannR. J.2002; Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans -complementation of Erns or E2 of CSFV. Vaccine 20:1544–1556[CrossRef]
van OirschotJ. T.,
KaashoekM. J.,
RijsewijkF. A.,
StegemanJ. A.1996; The use of marker vaccines in eradication of herpesviruses. J Biotechnol 44:75–81[CrossRef]
WalshE. P.,
BaronM. D.,
RennieL. F.,
AndersonJ.,
BarrettT.2000a; Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein. J Gen Virol 81:709–718
WalshE. P.,
BaronM. D.,
RennieL. F.,
MonaghanP.,
AndersonJ.,
BarrettT.2000b; Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers: evidence of exclusion of marker protein from the virus envelope. J Virol 74:10165–10175[CrossRef]
WasilkA.,
CallahanJ.,
Christopher-HenningsJ.,
GayB. T.,
FangY.,
DammenM.,
ReosM. E.,
TorremorellM.,
PolsonD.other authors2004; Detection of U.S. and Lelystad/European-like porcine reproductive and respiratory syndrome virus and relative quantitation in boar semen and serum by real-time PCR. J Clin Microbiol 42:4453–4461[CrossRef]
WassenaarA. L.,
SpaanW. J.,
GorbalenyaA. E.,
SnijderE. J.1997; Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that Nsp2 acts as a cofactor for the Nsp4 serine protease. J Virol 71:9313–9322
WidjojoatmodjoM. N.,
van GennipH. G.,
BoumaA.,
van RijnP. A.,
MoormannR. J.2000; Classical swine fever virus Erns deletion mutants: trans -complementation and potential use as non transmissible, modified, live-attenuated marker vaccines. J Virol 74:2973–2980[CrossRef]
WuW. H.,
FangY.,
FarwellR.,
Steffen-BienM.,
RowlandR. R. R.,
Christopher-HenningsJ.,
NelsonE. A.2001; A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF 2b. Virology 287:183–191[CrossRef]
WuW. H.,
FangY.,
RowlandR. R. R.,
LawsonS. R.,
Christopher-HenningsJ.,
YoonK. J.,
NelsonE. A.2005; The 2b protein as a minor structural component of PRRSV. Virus Res 114:177–181[CrossRef]
YuanS.,
MurtaughM. P.,
SchumannF. A.,
MickelsonD.,
FaabergK. S.2004; Characterization of heteroclite subgenomic RNAs associated with PRRSV infection. Virus Res 105:75–87[CrossRef]
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