veterinary sciences Review Challenges for Porcine Reproductive and Respiratory Syndrome (PRRS) Vaccine Design: Reviewing Virus Glycoprotein Interactions with CD163 and Targets of Virus Neutralization Ana M. M. Stoian and Raymond R. R. Rowland * Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506, USA; [email protected] * Corresponding Author: [email protected]; Tel.: 785-532-4631 Received: 19 December 2018; Accepted: 14 January 2019; Published: 17 January 2019 Abstract: One of the main participants associated with the onset and maintenance of the porcine respiratory disease complex (PRDC) syndrome is porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus that has plagued the swine industry for 30 years. The development of effective PRRS vaccines, which deviate from live virus designs, would be an important step towards the control of PRRS. Potential vaccine antigens are found in the five surface proteins of the virus, which form covalent and multiple noncovalent interactions and possess hypervariable epitopes. Consequences of this complex surface structure include antigenic variability and escape from immunity, thus presenting challenges in the development of new vaccines capable of generating broadly sterilizing immunity. One potential vaccine target is the induction of antibody that disrupts the interaction between the macrophage CD163 receptor and the GP2, GP3, and GP4 heterotrimer that protrudes from the surface of the virion. Studies to understand this interaction by mapping mutations that appear following the escape of virus from neutralizing antibody identify the ectodomain regions of GP5 and M as important immune sites. As a target for antibody, GP5 possesses a conserved epitope flanked by N-glycosylation sites and hypervariable regions, a pattern of conserved epitopes shared by other viruses. Resolving this apparent conundrum is needed to advance PRRS vaccine development. Keywords: porcine reproductive and respiratory syndrome virus; PRRSV; CD163; GP4; GP5; neutralizing antibody 1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) consists of two species: PRRSV-1 isolates are of European origin while PRRSV-2 originated in North America. PRRSV-1 and PRRSV-2 share ~60% identity at the nucleotide level. In addition, each species can be further divided into several clades or substrains. PRRSV, along with the closely related mouse virus, lactate dehydrogenase-elevating virus (LDV), are part of the Arteriviridae family, Variarterivirinae subfamily [1]. Further on, the two PRRSV species have been placed in genus Betaarterivirus, with Ampobartevirus and Eurpobartevirus as corresponding subgenuses. Subsequently, Betaarterivirus suid 1 species was created for the original PRRSV-1 and Betaarterivirus suid 2 species for the PRRSV-2, respectively. The progenitor virus for PRRSV-1 likely emerged in Eastern Europe and Russia followed by the introduction of PRRSV into North America, perhaps through the introduction of Russian wild boar [2]. A separate course of evolution in North America produced PRRSV-2. Perhaps the most remarkable aspect of PRRS virus evolution is the simultaneous emergence of PRRSV-1 and PRRSV-2, which produce similar disease signs and possess a similar epidemiology/ecology. Therefore, PRRSV is Vet. Sci. 2019, 6, 9; doi:10.3390/vetsci6010009 www.mdpi.com/journal/vetsci Vet.Vet. Sci. Sci.2019 2018, 6,, 95, x FOR PEER REVIEW 2 of 211 of 12 evolution is the simultaneous emergence of PRRSV-1 and PRRSV-2, which produce similar disease a goodsigns exampleand possess of howa similar a virus epidemiology/ecology. with unique biological Therefore, properties PRRSV is is able a good to effectively example of exploit how a virus unique ecologicalwith unique niches biological created properties by a modern is able swine to effect industry.ively exploit unique ecological niches created by a 0 modernThe PRRSV swine industry. genome possesses at least ten open reading frames (ORFs) flanked by a 5 leader and 30 untranslatedThe PRRSV region genome followed possesses by poly-Aat least ten tail. open The read nonstructuraling frames (ORFs) proteins, flanked encoded by a 5’ by leader ORF1a and and ORF1b,3’ untranslated possess protease, region followed replicase by andpoly-A host tail. gene The modulation nonstructural functions. proteins, Theencoded 30 end by of ORF1a the genome and codesORF1b, for atpossess least eightprotease, structural replicase proteins and host translated gene mo fromdulation a nested functions. 3’-coterminal The 3’ end set of of the subgenomic genome mRNAscodes for possessing at least eight a common structural leader, proteins a hallmarktranslated featurefrom a nested of the 3'-coterminal genus and the set Nidovirusof subgenomic order. ThemRNAs major structuralpossessing proteins, a common GP5, leader, matrix a hallmark (M), and feature nucleocapsid of the genus (N) are and encoded the Nidovirus by ORFs order. 5, 6, The and 7, respectively.major structural GP5 andproteins, M generally GP5, matrix exist (M), as aand GP5-M nucleocapsid heterodimer; (N) are however, encoded GP5 by ORFs homodimers 5, 6, and have7, beenrespectively. identified GP5 [3]. and GP2, M GP3, generally and GP4 exist are as minora GP5- surfaceM heterodimer; glycoproteins however, (GPs) GP5 derived homodimers from ORFshave 2, 3,been and 4,identified respectively. [3]. GP2, Two GP3, very and small GP4 nonglycosylatedare minor surface proteins—2bglycoproteins (or(GPs) E) derived and 5a—are from translatedORFs 2, 3, and 4, respectively. Two very small nonglycosylated proteins—2b (or E) and 5a—are translated from ORF2b and ORF5a, respectively [4,5]. In 2013, Kappes et al. [6] described the association of the from ORF2b and ORF5a, respectively [4,5]. In 2013, Kappes et al. [6] described the association of the nonstructural protein, nsp2, with the virion. However, there are no published data demonstrating that nonstructural protein, nsp2, with the virion. However, there are no published data demonstrating anti-nsp2 antibodies possess neutralizing activity. that anti-nsp2 antibodies possess neutralizing activity. The topological features of the virion surface are described in Spilman et al. [7], who performed The topological features of the virion surface are described in Spilman et al. [7], who performed cryo-electroncryo-electron microscopy microscopy followed followed byby tomographictomographic re reconstructionconstruction of of purified purified virions virions derived derived from from MARC-145MARC-145 cells cells infected infected with with aa PRRSV-2PRRSV-2 isolate.isolate. The The surface surface of of the the virion virion is issmooth, smooth, reflecting reflecting the the predominancepredominance of of the the short short peptide peptide sequencessequences formedformed by by the the ectodomains ectodomains of of M M and and GP5. GP5. A Asmall small numbernumber of of protrusions protrusions riserise above the the surface, surface, formed formed by bythe thelarge large ectodomains ectodomains of GP2, of 3, GP2, and 3,4. The and 4. Theectodomain ectodomain regions regions of surface of surface protei proteinsns are illustrated are illustrated in Figure in Figure 1. 1. FigureFigure 1. 1.Representation Representation of of porcineporcine reproductivereproductive and and respiratory respiratory syndro syndromeme virus-2 virus-2 (PRRSV-2) (PRRSV-2) virion virion surfacesurface proteins. proteins. The The proteins proteins are shownare shown for a representative for a representative PRRSV-2 PRRSV-2 isolate. The isolate. minor The glycoproteins minor GP2–4glycoproteins form a heterotrimer GP2–4 form protruding a heterotrimer from theprotrudi virionng surface. from the The virion surface surface. is dominated The surface by GP5-M is heterodimers.dominated by The GP5-M M protein heterodimers. is nonglycosylated. The M protei Then position is nonglycosylated. of the glycosylation The position sites (circles) of the for GP2–5glycosylation are from Dassites et(circles) al. and for Ansari GP2– et5 are al. [from8,9]. AsterisksDas et al. showand Ansari those et N-sites al. [8,9]. required Asterisks for show infection those [8 ,9]. TheN-sites dashed required line identifies for infection the disulfide [8,9]. The bond dashed between line identifies GP5 and the M. disulfide The structures bond between are notdrawn GP5 and to M. scale. The structures are not drawn to scale. The targets for PRRSV infection are cells of monocyte/macrophage origin. It is this interaction betweenThe the targets virus andfor PRRSV macrophage infection that are is responsiblecells of monocyte/macrophage for respiratory distress origin. and It immune is this interaction modulation, whichbetween are associated the virus with and the macrophage onset of PRDC. that Vanis re Breedamsponsible et al.for [ 10respiratory] were the firstdistress to propose and immune a detailed modelmodulation, describing which how are PRRSV associated interacts with the with, onset and of then PRDC. enters, Van theBreedam macrophage et al. [10] host. were In the this first model, to PRRSVpropose infection a detailed occurs model in describing three steps, how which PRRSV incorporate interacts with, interactions and then between enters, the PRRSV macrophage and three differenthost. In receptorthis model, molecules PRRSV oninfection the macrophage occurs in three cell steps, surface. which The incorporate first step is interactions the initial interactionbetween PRRSV and three different receptor molecules