Antiviral Strategies against PRRSV Infection

Antiviral Strategies against PRRSV Infection

TIMI 1473 No. of Pages 12 Review Antiviral Strategies against PRRSV Infection Taofeng Du,1,2,3 Yuchen Nan,1,2,3 Shuqi Xiao,1,2 Qin Zhao,1,2 and En-M...

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TIMI 1473 No. of Pages 12

Review

Antiviral Strategies against PRRSV Infection Taofeng Du,1,2,3 Yuchen Nan,1,2,3 Shuqi Xiao,1,2 Qin Zhao,1,2 and En-Min Zhou1,2,* [390_TD$IF]PRRSV (porcine reproductive and respiratory syndrome virus) is a major economically significant pathogen that has adversely impacted the global swine industry for almost 30 years. Currently PRRSV is estimated to cause losses of almost US$600 million per year in the USA. Except for new mutants that continually emerge during PRRSV outbreaks, our understanding of the virology, origin, and evolution of PRRSV and the host's immune response are largely inadequate. Such limited knowledge impedes development of effective methods to eradicate this virus. In this review, we systematically describe recent advances in anti-PRRSV research, especially focusing on those techniques with the potential to transform current anti-PRRSV strategies. Furthermore, a combination of these new techniques may provide creative insights to guide future PRRSV control and prevention.

Trends Since the first report of a PRRS outbreak, and the launch of the first modified live-virus vaccine decades ago, tremendous efforts have been made to control this disease. However, it is still one of the most economically significant diseases impacting the swine industry globally. PRRSV constantly evolves to cause new outbreaks by evading the existing immunity in vaccinated herds. Consequently, atypical virulent strains are frequently isolated from new outbreaks. Our understanding of PRRSV manipulation of the host immune system is still inadequate, and current antiPRRSV strategies are ineffective and impractical for controlling PRRSV.

PRRSV Infection in Swine The genome of PRRSV (porcine reproductive and respiratory syndrome virus; see Glossary) is approximately 15 kb in size and is organized with replicase genes located at the 5ʹ end and genes encoding structural proteins comprising the remainder of the sequence [1]. There are two well known PRRSV genotypes: type 1, or European-like (prototype Lelystad), and type 2, or North American-like (prototype VR-2332) [2]. These two genotypes share approximately 60% sequence identity and exhibit serotype differences [1]. Unlike other members of the genus Arterivirus which exhibit relatively broad cell tropism [3], PRRSV infection is highly restricted to cells of the monocyte–macrophage lineage such as [391_TD$IF]porcine alveolar macrophages (PAMs), the primary targets of PRRSV in vivo [1]. Since the identification and characterization of PRRSV, new virulent PRRSV strains have constantly evolved and caused new outbreaks around the world (Box 1). The typical immune features of PRRSV infection in the host include persistence viremia, a strong inhibition of innate cytokines (IFN-a/b, TNF-a, IL-1b etc.), dysregulation of NK cell function, rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibody, a late and low CD8+[389_TD$IF] T-cell response, and induction of regulatory T cells (Tregs) (for details see [1]). To prevent PRRSV infection, a [392_TD$IF]modified live vaccine (MLV, Ingelvac PRRS1 MLV) has been commercially available for more than two decades. Recently, multiple MLV [39_TD$IF]against both genotypes have also been developed. However, the prevalence of PRRSV infection in swine (Sus domesticus) herds is still high, and vaccination has achieved little success [4] (Box 2). The dilemma of PRRSV control has been somewhat surprising, since a number of vaccines against [394_TD$IF]equine arteritis virus[395_TD$IF] (EAV), another member of the genus Arterivirus, are available and effective [5]. Despite the sustained effort, PRRSV-specific treatment for infected herds, or prevention methods other than vaccines, are still unavailable. Moreover, except for the high mortality

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Combined with new technical advances in recent years, certain novel anti-PRRSV methods have been proposed that demonstrate the potential to transform our ability to control PRRSV.

1

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China 2 Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, Shaanxi, China 3 These authors contributed equally to this work *Correspondence: [email protected] (E.-M. Zhou).

http://dx.doi.org/10.1016/j.tim.2017.06.001 © 2017 Elsevier Ltd. All rights reserved.

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Box 1. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Constantly Evolves to Cause New Outbreaks Initial outbreaks of PRRS were reported simultaneously in North America (late 1980s) and Western Europe (1990s) [1]. Since then, this disease has quickly spread to the rest of the world [1], and new virulent variants of PRRSV have constantly evolved and caused new outbreaks globally. In the late 1990s, an atypical PRRSV strain causing high mortality and abortion in vaccinated herds was reported in the USA [368_TD$IF][94]. Since 2001, many virulent isolates belonging to the same group of viruses (with a common 1-8-4 restriction fragment length polymorphism pattern) emerged as early cases in Canada and in Minnesota, USA. These studies led to the discovery of a highly virulent MN184 strain which is quite distinct (>14.5% nucleotide dissimilarity) from other genotype 2 PRRSVs [369_TD$IF][95]. In 2006, a new type of highly pathogenic PRRSV (now referred to as HP-PRRSV) was identified that caused high mortality (20–100%) in sows in South China and in North Vietnam [370_TD$IF][96]. HP-PRRSV has since spread to Southeast Asia and India [371_TD$IF][97,98]. Meanwhile, identification of a new genotype 2 strain, NADC30, in the US in 2008 preceded isolation of NADC30-like strains (with mortality rates of 30–50%) in central China as well [372_TD$IF][99]. Notably, after large-scale vaccination using a MLV[37_TD$IF], field isolates from a PRRSV outbreak exhibited nearly identical nucleotide sequences to vaccine strains, with recombination between MLV and wild-type strains also reported [374_TD$IF][100–103]. Unfortunately, it appears that PRRSV is constantly evolving by circumventing host immunity; consequently, outbreaks caused by new variants may never end.

and morbidity, PRRSV infection causes hosts to be more susceptible to secondary infection by other viruses or bacteria. In the field, various management procedures have been implemented to achieve farm and regional control of PRRSV infection; these include a PRRSV test for semen and gilt acclimation, removal of the seropositive animal, herd depopulation and repopulation, and herd closure and rollover; however, control and elimination of PRRSV within a relatively large region is much more complicated and is expected to require a much longer-term commitment [6]. Therefore, effective PRRSV control and prevention methods are urgently needed. In this review, we summarize recent advances for the development of anti-PRRSV interventions based on diverse concepts (Figure 1, Key Figure). In this way, we hope to provide creative insight and vision to guide development of innovative strategies to achieve PRRS prevention and control.

PRRSV Entry Blockers PRRSV has a highly restricted cell tropism both in vitro and in vivo. Numerous studies have demonstrated that PRRSV infection is dependent on various cellular receptors or factors, such as heparin sulfate (HS), vimentin, CD151, CD163, sialoadhesin (CD169, Sn), DC-SIGN (CD209) and non-muscle myosin heavy chain 9 (NMHC II-A or MYH9) [1,7]. However, recent reports have demonstrated that CD163 is indispensable for mediating PRRSV infection both in vitro and in vivo [8]. CD163 knockout pigs are resistant to PRRSV infection [9], and the soluble form of truncated CD163 (containing domains SRCR5–SRCR9) and Sn are able to capture PRRSV particles and inhibit virus replication in vitro. The SRCR5 domain of CD163 appears to Box 2. Current Licensed Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccines Are Unreliable and Ineffective Since the discovery of PRRSV, several attenuated vaccines have been developed against both genotypes and licensed in various countries according to their particular circulating genotypes. Porcilis1 PRRS (Merck), Ingelvac PRRSFLEX EU (Boehringer Ingelheim), Amervac-PRRS (Hipra), Pyrsvac-183 (Syva), and ReproCyc PRRS EU (Boehringer Ingelheim) were developed against genotype 1 PRRSV and are mainly licensed in West European countries. Ingelvac PRRS1 MLV (Boehringer Ingelheim), ReproCyc PRRS-PLE (Boehringer Ingelheim), and Ingelvac PRRS1ATP (Boehringer Ingelheim) were developed against genotype 2 PRRSV and are mainly licensed in the USA and China. No efficacy data have yet been obtained for Ingelvac PRRSFLEX EU and ReproCyc PRRS EU, both launched in 2015. Previously licensed PRRS MLV of either genotype 1 or genotype 2 did elicit relatively weak humoral and cell-mediated immune responses, as seen for virulent strains [376_TD$IF][104]. It appears that vaccines confer late but effective protection against genetically homologous PRRSV, but only partial protection against heterologous strains [376_TD$IF][104]. This conclusion is also consistent with data for reported atypical PRRS outbreaks detected in vaccinated herds since 1996 [37_TD$IF][94,105]. Ingelvac PRRS1[375_TD$IF] MLV was the first licensed PRRSV vaccine and was widely administered in both China and the USA. Subsequently, researchers from both countries have collected field PRRSV isolates from outbreaks which exhibit nearly identical nucleotide sequences to Ingelvac PRRS1 MLV [100,101]. Moreover, recombination between vaccine and wild-type strains has been identified as well [98]. Therefore, practitioners in the swine industry are concerned not only about MLV [379_TD$IF]efficacy, but also about the safety of current attenuated vaccines generated using similar means.

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Glossary CD151: Cluster of Differentiation 151, a member of the transmembrane 4 superfamily, mediates cell signal transduction and is characterized by the presence of four hydrophobic domains. CD163: Cluster of Differentiation 163, the high-affinity scavenger receptor for the hemoglobin– haptoglobin complex, indispensable for PRRSV infection and also a receptor for simian hemorrhagic fever virus, another member of Arterivirus. CD169: Cluster of Differentiation 169, also known as sialoadhesin, an I-type lectin and cell-adhesion molecule that binds to sialic acids and is expressed in macrophages. CD209: Cluster of Differentiation 209, also known as dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin, a C-type lectin receptor present on the surface of both macrophages and dendritic cells. CL907: a TLR-7/8 agonist. EAV: equine arteritis virus, belonging to the genus Arterivirus, infects horses and causes fever, edema, conjunctivitis and abortion. GP: glycoprotein, a type of protein containing oligosaccharide chains covalently attached to peptide sidechains. They are present on the surface of viral envelopes and serve as ligands for binding to host cell receptors. They are often major targets for antibody- mediated virus neutralization. HO-1: heme oxygenase-1, a ubiquitously expressed inducible isoform of the rate-limiting enzyme for heme degradation. It produces biliverdin, ferrous iron, and carbon monoxide. IFN: interferon, large families of genetically and functionally related proteins that interfere with virus replication and play key roles in host innate immunity. LDV: lactate dehydrogenaseelevating virus, another member of Arterivirus, infects mice. MARC-145: a cell line used widely for PRRSV propagation, derived from the African green monkey kidney cell line MA104. MicroRNA (miRNA): evolutionarily conserved, small non-coding RNA molecules (containing about 22 nucleotides) found in plants, animals, and some viruses. miRNA functions in RNA silencing and post-

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be essential for PRRSV infection since antibody recognizing CD163-SRCR5 can block PRRSV infection in vitro [10]. However, the SRCR5 domain [396_TD$IF]is not sufficient since the purified SRCR5 domain is unable to capture PRRSV virions [11]. Based on idiotype network theory, a mouse (Mus musculus) monoclonal anti-idiotypic antibody (Mab2-5G2), specific for idiotypic antibodies that recognize PRRSV GP5, was shown to recognize MYH9, which is ubiquitously expressed in eukaryotic cells. MYH9 C-terminal domain acts as an essential factor involved in PRRSV infection through its physical interaction with the GP5 protein [7,12]. Mab2-5G2 treatment or intracellular expression of the Mab2-5G2 scFV region in PRRSV-susceptible cells significantly inhibits PRRSV replication [13]. Moreover, blebbistatin, a myosin II ATPase inhibitor [14], blocks PRRSV replication both in vitro and in vivo [7]. Taken together, truncated CD163, Mab2-5G2 scFV, and blebbistatin [397_TD$IF]target crucial steps in PRRSV [398_TD$IF]viral entry to block PRRSV entry. Therefore, PRRSV entry blockers are a promising novel approach to combat PRRSV infection.

The Proviral and Antiviral Role during PRRSV Replication by Host MicroRNAs MicroRNAs [39_TD$IF](miRNAs) have recently emerged as key regulators that play important roles during viral replication [15]. The function of miRNAs may be either proviral or antiviral [15]. The miRNAs involved in modulating PRRSV replication can be classified into three different categories depending on their targets: (i) direct targeting of the PRRSV genome; (ii) targeting signaling pathways involved in PRRSV replication; and (iii) targeting host factors involved in PRRSV replication. miRNAs Targeting the PRRSV Genome Targeting of viral genomes by host miRNAs has been documented for both DNA and RNA viruses infecting mammalian species and is a newly discovered host antiviral defense mechanism [15]. Research on other viruses has indicated that certain miRNAs determine cell susceptibility to virus and have a significant effect on viral evolution and tissue tropism [15]. The first identified PRRSV genome-targeting miRNA was miRNA-181, which inhibits genotype 2 PRRSV replication by binding to a highly conserved region downstream of open reading frame (ORF) 4 [16][40_TD$IF]. The details of PRRSV genome-targeting miRNAs identified to date, and their targets, are summarized in Table 1. It is interesting that the expression of miRNA-181 and several other miRNAs is inversely correlated with PRRSV infectivity in different cells and tissues [16]. This observation indicates that these PRRSV-targeting miRNAs might potentially and significantly influence tissue tropism or even host tropism.

transcriptional regulation of gene expression. MLV: modified live vaccine, a type of vaccine produced by disabling virulent properties of the parent pathogen which results in virus which no longer causes disease in susceptible hosts. Nanobody: derived from the variable domains of Camellidae heavy chainonly antibodies (VHH). Nanobodies possess many attractive features, such as small size, ease of genetic manipulation, high specificity, and solubility. PAM: porcine alveolar macrophage, a type of macrophage found in pulmonary alveoli. PRRSV: porcine reproductive and respiratory syndrome virus, an enveloped positive-stranded RNA virus belonging to the genus Arterivirus, family Arteriviridae and order Nidovirales. SRCR: scavenger receptor cysteinerich protein domain, present on the cell membrane, binds to specific ligands and is often involved in immune system function. TLRs: Toll-like receptors, a receptor family most closely related to Drosophila toll protein homologs; they serve as receptors for pathogen-associated molecular patterns and play key roles in host innate immunity. Treg: regulatory T cells, a subpopulation of T cells which modulate immune system function and maintain tolerance to selfantigens for the prevention of autoimmune disease.

Table 1. List of miRNAs That Have Anti-PRRSV Properties miRNA No.

Targets or signaling pathways

Refs

miRNA-23

ORF3 and type I interferon responses

[18]

miRNA-378

ORF7

[18]

miRNA-505

ORF3 and ORF5

[18]

miRNA-130

5ʹUTR

[32]

miRNA-181

CD163-3ʹUTR

[22]

let-7f-5p

NMHC-2A-3ʹUTR

[23]

miRNA-506

CD151-3ʹUTR

[24]

miR-26a

Type I interferon responses

[15]

miR-30c

Type I interferon responses

[16]

miR-125b

NF-kB signaling pathway

[17]

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Key Figure

Current Techniques Employed to Inhibit Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Replication at Multiple Steps. PRRSV parcles 7. Immune smulators

1.Viral entry blockers

PRRSV receptors Internalizaon

6. Downregulang host receptors

2. Acvang TLRs signaling

Endosome Uncoang

TLR3/7

(+) Genome Translaon of pp1a and pp1b

Budding (–) Genome

3.Translaon inhibion and genome degradaon

Golgi (–) sg mRNAs

(+) Genome

(+) sg mRNAs

RTC RTC

4.Enhancing type I IFN signaling

5.Blocking RTC assembly?

IRF3/7

Endoplasmic reculum

IFNs/cytokines Nucleus

Figure 1. 1. Viral entry blockers: truncated CD163, monoclonal anti-idiotypic antibody Mab2-5G2, blebbistatin, Mab2-5G2 scFV, and neutralizing antibodies. 2. Activating Toll-like receptors (TLRs) to induce type I IFN: poly(I:C), SZU101, and CR907 act as TLR ligands to [365_TD$IF]activate type I IFN response. 3. Translation inhibition and genome degradation: miRNA-23, miRNA-378, miRNA-505, miRNA-130, [36_TD$IF]peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), and siRNA and shRNA directly target the PRRSV genome. 4. Enhancing type I IFN signaling: miRNA-23, miR-26a, and miR-30c to enhance type I IFN signaling. 5. Blocking assembly of the RTC (replication and transcription complex): nanobodies targeting NSP4 and NSP9 may block them from engaging in the RTC assembly. 6. Downregulating host receptors: miRNA-181 and let-7f-5p targeting CD163-3ʹUTR and MYH9-3ʹUTR, respectively, to downregulate the receptor expression. 7. Immune stimulators: type I IFN and type II IFN evoke both innate and adaptive immunity against PRRSV infection.

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miRNAs Targeting Signaling Pathways Involved in PRRSV Replication miRNAs may exert additional effects to prevent PRRSV function. For example, miR-26a and miR-30c have been shown to inhibit PRRSV replication by inducing type I interferon responses [17,18]. Using a different target, miR-125b may act to reduce PRRSV replication by upregulating the NF-kB signaling pathway [19]. In addition to targeting the PRRSV genome, miRNA-23 also exhibits the ability to enhance type I IFN induction by the activation of IRF3/IRF7 during PRRSV infection; therefore, miRNA-23 exerts multiple inhibitory effects on PRRSV replication [20]. However, no specific targets for these miRNAs within these signaling pathways have yet been identified. Notably, certain miRNAs play a proviral role in PRRSV replication. For example, miRNA-373 impairs interferon (IFN)-b production by targeting nuclear factor I (NFI A, NFI B), interleukin-1 receptor-associated kinase (IRAK1, IRAK4) and interferon regulatory factor (IRF1) to facilitate PRRSV replication in vitro [21]. Alternatively, miR-29a promotes PRRSV replication during the early stage of virus infection by targeting AKT serine/threonine kinase 3 (AKT3) [22]. Furthermore, upregulated miR-30c during PRRSV infection is able to dampen signaling by type I IFNs by targeting Janus kinase 1 (JAK1), a tyrosine kinase protein essential for signaling for certain type I and type II cytokines, which promotes PRRSV infection [23]. miRNAs Targeting Host Factors Involved in PRRSV Replication Several miRNAs are able to target host factors involved in PRRSV replication. The PRRSV receptors CD163 and MYH9 appear to be targeted by miRNA-181 and miRNA let-7f-5p, respectively, to reduce PRRSV replication in vitro [24,25]. Moreover, host miRNA-506 targets coreceptor CD151 to block PRRSV replication within MARC-145 cells [26]. Our own studies have demonstrated that [401_TD$IF]heme oxygenase (HO-1, also known as Hsp32), is able to block PRRSV replication via its downstream metabolites carbon monoxide (CO) and biliverdin [27]; meanwhile, HO-1 itself is targeted by PRRSV-induced upregulation of miR-24-3p and miR-22 to promote PRRSV replication [28,29].

An Antisense RNA-Based Strategy [402_TD$IF]As a PRRSV-Specific Therapy With the exception of host miRNAs, other antisense-based strategies including small interfering RNAs (siRNAs), short-hairpin RNAs (shRNAs), artificial microRNAs, and PMOs (morpholino oligomers) have been tested for inhibition of PRRSV infection both in vitro and in vivo. The siRNAs targeting nonstructural protein (NSP) 1a [30], NSP9 [31], and N genes [32] of PRRSV had been reported to block PRRSV replication in permissive cell lines. In addition, an shRNA targeting the ORF1 region of PRRSV significantly suppressed PRRSV replication as well [33]. Moreover, in vivo studies demonstrated that intranasal inoculation of piglets with either miR-181c or miR-130b mimics exhibited strong anti-PRRSV effects [16,34]. In yet another example, recombinant pseudorabies virus expressing siRNAs against ORF7 of the highly pathogenic PRRSV (HP-PRRSV) strain HN1 inhibited virus replication and reduced gross lung lesions in piglets [35]. Finally, an alternative strategy utilizing RNAi has been used to produce transgenic pigs bearing PRRSV-specific shRNA to confer PRRSV resistance. However, stable expression of shRNA targeting the PRRSV-N protein in transgenic piglets extended their survival time by only 3 days compared with wild-type piglets upon HP-PPRSV challenge [36]. Thus, it appears that complete resistance to PRRSV may be difficult to achieve in transgenic pigs that only synthesize PRRSV-specific siRNA. It is possible that the major obstacle to siRNA and shRNA action in vivo is poor cellular uptake resulting from inadequate intracellular delivery methods, instability under physiological conditions, off-target effects, and nonspecific immune stimulation. Therefore, nuclease-resistant oligonucleotide analogues, such as PMOs, are being tested as another promising antisense strategy [37]. Published reports suggest that the use of PMO-5UP2, which contains sequence

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complementary to the conserved region in the 5ʹ-terminal end of the PRRSV genome, results in a multi-log10 inhibition of PRRSV replication as well as cross-strain protection against heterogeneous PRRSV isolates in vitro [37]. Moreover, intranasal introduction of PMO-5UP2 significantly reduced PRRSV viremia and PRRSV-induced interstitial pneumonia [38], suggesting that PMO-5UP2 holds promise as a novel PRRS control candidate.

Herbal Extracts, Chemical Compounds, and Nanobodies for PRRSV Inhibition Since traditional Chinese medicine (TCM)-based herbology has been widely used as a source of novel drugs, many crude TCM herbal extracts have been shown to inhibit PRRSV replication. Tanshinone IIA, one of the most abundant constituents in the root of Salvia miltiorrhiza, inhibits PRRSV by suppressing N gene expression and blocking virus-induced apoptosis [39]. Epigallocatechin gallate (EGCG), the most abundant catechin found in tea [40], was found to inhibit PRRSV replication in MARC-145 cells as well [41]. Tea seed saponins, present in crude extracts from tea, inhibited PRRSV in a similar manner as achieved using Tanshinone IIA [42]. Meanwhile, another in vitro study aimed at screening potential antiPRRSV compounds derived from herbs demonstrated that chlorogenic acid and scutellarin exhibited the highest potential anti-PRRSV activities [43]. Fungal compounds such as flavaspidic acid AB (FA-AB), a compound derived from Dryopteris crassirhizoma, exhibited anti-PRRSV activity through targeting of multiple stages of PRRSV infection in vitro, in addition to inducing IFN-a, IFN-b, and IL1-b expression in PAMs [44]. CM-H-5, another novel fungal compound isolated from the water-soluble fraction of the mushroom Cryptoporus volvatus, inhibits PRRSV replication as well [45]. Additional chemical compounds, such as tetrahydroaltersolanol C [46], N-acetylpenicillamine [47] and dipotassium glycyrrhetate, have been shown to inhibit PRRSV replication in vitro [48]. However, all of these studies only tested anti-PRRSV activity of crude extracts or agents in vitro and did not test efficacy in vivo. Moreover, the anti-PRRSV activity of these agents appears to be nonspecific since some of them appear to inhibit replication of multiple diverse viruses. The underlying mechanisms responsible for their antiviral activity are still elusive. Therefore, these agents are far from being ready for practical use in anti-PRRSV therapy. Nanobodies are single-chain antibody fragments derived from the variable domains of Camellidae heavy chain-only antibodies (VHH) which have binding specificity to several viruses and act in unique ways due to their small size, ease of genetic manipulation, high specificity, and solubility. For PRRSV, nanobodies targeting the NSP9 and NSP4 proteins were screened and tested for anti-PRRSV properties [49,50]. Two nanobody coding sequences were selected and intracellularly expressed by MARC-145 cells and were shown to inhibit PRRSV infection by potentially blocking these NSPs, two of the ten NSPs proteolytically processed from replicase polyproteins (pp1a and pp1b) [1], from engaging in the viral replication and transcription complex (RTC) assembly. Therefore, nanobodies hold great potential for development as novel antiviral treatments for PRRSV infection.

Immune Stimulators and Their Potential [402_TD$IF]As Adjuvants for PRRSV Vaccines The PRRSV genome encodes several IFN antagonists which block IFN production and signaling [1] (Box 3). One strategy for combating PRRS entails activating the innate immune response using Toll-like receptor (TLR) agonists. Several TLR ligands, including poly (I:C), SZU-101, and CL907, which are recognized by TLR3, TLR7, and TLR7/8, respectively, are able to inhibit PRRSV replication in vitro [51–56]. In mouse models, activation of TLR7 combined with immunization of inactive PRRSV vaccine induced high levels of PRRSV-specific humoral immune responses and T lymphocyte proliferation [54]. Similar results were observed when swine were immunized with inactive vaccine with TLR3 and TLR7/8 ligands [55].

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Box 3. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Is Capable of Evading Type I Interferon-Mediated Antiviral Response Recent reports show that the PRRSV genome encodes several IFN (interferon) antagonists which block either IFN induction or IFN-activated JAK/STAT signaling [380_TD$IF][106–108]. The nsp1 of PRRSV generates two self-cleaved subunits: nsp1a and nsp1b [381_TD$IF][109]. Both of them dramatically inhibit IFN-b expression by affecting IRF3-mediated IFN induction [381_TD$IF][109]. The nsp2, the largest nonstructural protein encoded by PRRSV, antagonizes IFN induction by blocking IRF3 phosphorylation and nuclear translocation [382_TD$IF][110,111]. Moreover, nsp4 is another IFN antagonist which interferes with the NF-kB signaling pathway through the cleavage of NEMO to dampen IFN-b induced by poly (I:C) [38_TD$IF][112]. Furthermore, nsp11 is able to suppress the activation of IFN-b by cleaving the mRNA of IPS-1 via the endoribonuclease domain [384_TD$IF][113]. Additionally, other than nsps of PRRSV, structural proteins, such as N protein, prevent IFN-b induction in a manner similar to that of nsp2 [385_TD$IF][114]. Except for inhibiting IFN induction, PRRSV nsp1b prevents IFN-activated JAK/STAT signaling via promoting degradation of KPNA1, a key transporter for mediating the nuclear import of ISGF3 [386_TD$IF][106,115]. Moreover, nsp7, nsp12, GP3, and N of PRRSV also interfere with IFN-activated signaling by an unknown mechanism [387_TD$IF][61,108].

Since activation of TLRs leads to induction of IFNs and other cytokines [57], a variety of IFN types have been tested for their anti-PRRSV activity or adjuvant potential both in vitro and in vivo. Recombinant swine IFN-b and IFN-g inhibit PRRSV replication in MARC-145 cells and PAMs [58–60]. However, the antiviral activity of IFN-b is strain-dependent in MARC-145 cells, which is consistent with observations regarding the effects of PRRSV strain differences on inhibition of IFN-activated JAK/STAT signaling [61]. In vivo, pretreatment of swine with IFN-a prior to challenge eased PRRSV-induced symptoms. However, it appears that IFN therapy was unable to rescue PRRSV-infected swine from death, but it did extend survival time [62]. Type III IFNs, a newly defined IFN family, also have anti-PRRSV activity in MARC-145 cells [63]. Meanwhile, as an endogenous IFN-g (type II IFN) inducer, recombinant porcine IL-12 has been demonstrated to block PRRSV replication in PAMs [64]. Since type II IFNs play a central role in cell-mediated immunity, recombinant porcine IL-12 was tested and shown to enhance humoral and cell-mediated immune responses in swine when used as an adjuvant with an inactivated PRRSV vaccine [65]. However, when combined with MLV, IL-12 coimmunization increased cell-mediated but not humoral immune responses, leading to only partial improvement of MLV efficacy [66]. In a different strategy, coadministration of plasmid DNA encoding IFN-g with either PRRSV-ORF5 or ORF7 DNA vaccines reduced virus load and lung lesions in swine after PRRSV challenge [67]. Meanwhile, swine IL-2 expressed by a eukaryotic expression plasmid effectively strengthened humoral and cell-mediated immune responses to a DNA vaccine carrying PRRSV ORF5, ORF7, and ORF3/ORF5 [68,69]. In addition to interference with innate immunity, a primary cause of immunosuppressive and persistent infection of PRRSV is linked to upregulation of IL-10 and induction of Tregs during infection [1]. Plasmids encoding short hairpin RNAs to block IL-10 mRNA expression exhibited increased proinflammatory cytokine production after PRRSV infection in naive porcine peripheral blood mononuclear cells [70]. From current knowledge of immune-modulatory roles of Tregs and IL-10 in PRRSV infection, Tregs and IL-10 hold promise as targets for PRRSV control, but this idea awaits further investigation. Recently, two PRRSV strains with unique abilities to induce type I IFNs production have been documented [71,72]. One of them, PRRSV-A2MC2, a moderately virulent strain, shares the highest identity with prototype strain VR-2332 and is the first reported strain with the ability to induce IFN synthesis [73]. In vivo studies demonstrated enhanced neutralizing antibody production in A2MC2-infected swine compared with swine infected with MLV or virulent VR-2385 strains [73]. In heterogeneous PRRSV challenge experiments, attenuated A2MC2 strains after 90 passages in MARC-145 cells were able to protect pigs against challenge with VR-2385 (with 92.3% nucleic acid identity to A2MC2) but not against challenge with the highly

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virulent strain MN184 (with 84.5% nucleic acid identity to A2MC2) [74]. Taken together, these data suggest that attenuated PRRSV strains maintaining IFN-inducing phenotype are more likely to elicit adaptive immunity during immunization which may be superior to inactive or DNA vaccines that require TLR ligands or recombinant cytokines as adjuvants for immunization.

PRRSV Neutralizing Targets and Implications for Future PRRSV Vaccines Although antibody responses during PRRSV infection were initially viewed as a deleterious and ineffective component of PRRSV-specific immunity, neutralizing antibodies are now considered to be essential to combat PRRSV [1]. In experimental infection, the onset of neutralizing antibody is accompanied by virus clearance [75]. In addition to the fact that genetic and antigenic variability among PRRSV isolates has hampered effective prevention and control using an antibody-based strategy [1], the major neutralizing targets among PRRSV antigens are still controversial. Initially, the major glycosylated envelope protein (GP5), encoded by PRRSV-ORF5, was proposed to be the primary target for neutralization, as observed for its counterparts in [403_TD$IF]lactate dehydrogenase-elevating virus[395_TD$IF] (LDV) and equine arteritis virus (EAV) [76]. The importance of GP5 is also supported by the identification of a neutralizing epitope (the B epitope, amino acids 37–45 of GP5) and the discovery of neutralization-resistant mutants containing amino acid substitutions in GP5 [77–79]. Conversely, emerging evidence continually challenges the assertion that GP5 is the sole or a major neutralizing target accounting for the universal neutralization of heterogeneous isolates. First, reports have indicated that monoclonal antibodies recognizing the putative ‘major neutralizing epitope' located in GP5 demonstrated little neutralizing activity [80]. Moreover, since PRRSV GP5 and M form a heterodimer via a disulfide bond, another study tested PRRSV M-GP5 ectodomain-specific antibodies from PRRSV-neutralizing serum. While these antibodies bound virus, they did not achieve virus neutralization [81]. Conversely, in the European prototype strain Lelystad, peptide scanning suggested that E, GP5, or M were not targeted by virus-neutralizing antibodies, while GP3 appears to be the major neutralizing target based on sera collected from Lelystad-infected swine [82]. Meanwhile, available data also support PRRSV-GP4 as the viral-neutralizing target in both genotypes, and it has been proposed as a driving force for PRRSV evolution [83–85]. Adding to the controversy, neutralizing epitopes from GP2 have been reported as well [82]. To date, the mechanism of antibody-mediated PRRSV neutralization is still elusive, since conflicting data have been obtained by various research groups. One plausible explanation for these discordant results is that antigenic determinants and their biological properties may differ radically among the homologous versus heterologous PRRSV isolates used in the various studies [86]. With regard to the heterologous nature of PRRSV neutralization, B epitopes of GP5 (a well-recognized neutralization epitope of VR-2332) do not play a role in neutralization of the HP-PRRSV-HuN4 strain; the only amino acid mutation in the B epitope of HuN4-GP5 is not predicted to be involved in antibody recognition [87]. Therefore, cross-protection of MLVs based on a single strain against heterologous strains is always poor [1]. Currently, research is focused on swapping genetic segments encoding structural proteins from heterologous PRRSVs as a new strategy for designing the next generation of vaccines. Promising data have demonstrated that shuffling GP4 and M between strains to generate chimeric virus may broaden the heterologous cross-neutralizing antibodies that are induced [88]. A recent study demonstrated that, by shuffling structural genes (ORFs 3-6) from six heterologous PRRSV strains and inserting them into a PRRSV strain VR-2385-based backbone, the rescued chimeric virus exhibited improved cross-protective efficacy against multiple

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heterologous strains [71]. Moreover, a synthetic strain (but still virulent for swine) based on the consensus genome created by aligning 59 full PRRSV genomes achieved unprecedented levels of heterologous protection [89]. In contrast to [40_TD$IF]MLVs, inactivated PRRSV vaccines have been developed and licensed world wide. Although the performance of inactivated vaccine against wild-type PRRSV infection is questionable [90], long-term administration of inactivated vaccine in seropositive herds demonstrates a significant improvement in sow reproductive performance [91], suggesting the potential of inactivated PRRSV vaccines as therapeutic or antiviral agents if the identification of neutralization targets and heterogeneous protection of PRRSV can be further explored. In contrast to antibodies, our understanding of PRRSV envelope antigens is largely limited. Indeed, since the techniques employed for epitope mapping have relied heavily on synthesized peptides or mouse monoclonal antibodies, [405_TD$IF]they ignore the existence of conserved conformational epitopes. Evidence of HIV broadly neutralizing antibodies produced by HIV-specific B cells further suggests that such antibodies may similarly exist in PRRSV naturally infected hosts [92]; this supposition is consistent with the recent discovery of pig sera containing broadly neutralizing antibodies against both PRRSV genotypes [75]. If the conserved epitopes recognized by broadly specific neutralizing antibodies could be further characterized, this information could promote development of a cross-protective vaccine against heterogeneous PRRSV strains.

Concluding Remarks It has been several decades since the emergence of PRRSV in 1987. Unfortunately, a sustained effort dedicated to understanding PRRSV pathogenesis, and the development of improved vaccines, has achieved only limited success (Box 2, and see Outstanding Questions). On the one hand, although gene-editing of CD163 suggests a potential way to develop PRRSVresistant pigs, it is still far from being in agricultural use, especially with regard to the poor acceptance of GMOs (genetically modified organisms) as food sources in most countries. On the other hand, identification of both anti- or pro-PRRSV miRNAs offers alternative targets for gene editing, but faces the same dilemma. Although vaccination for food animals is still a common approach to control infectious diseases, vaccination against PRRSV infection has achieved little success. Antiviral treatment for food animals has not been attempted, probably because of the under-developed technology and for economic reasons. However, the antiviral strategies against PRRSV infection summarized in this review could be useful for boar studs and sows to block semen-mediated PRRSV transmission and persistent infection of piglets survived in utero, which will effectively eliminate PRRSV circulation within a naïve herd [93]. As for smallpox in humans and rinderpest in cattle, pigs (except wild boar) are the sole natural host species for PRRSV, with no evidence of cross-species transmission. Therefore, as previously achieved for smallpox and rinderpest, global campaigns utilizing large-scale immunization with effective vaccines may quickly eradiate this pathogen from swine herds worldwide. Currently, two novel strategies hold great promise for the development of such a vaccine. One strategy involves artificially swapping or shuffling genetic elements to create crossprotective chimeric virus vaccines. The second entails the use of IFN-inducible strains to restore the host immune response. Both strategies show promise when compared with conventional MLVs based on single strains. Moreover, inactivated PRRSV vaccine as a therapeutic or antiviral agent for PRRSV-positive herds is promising as well. Hopefully, novel PRRSV vaccines based on the above techniques will be available in the near future and will prove to be highly effective tools against PRRSV.

Outstanding Questions What determines host tropism for PRRSV? In vitro culture systems of PRRSV are limited to PAMs and MARC-145 cells. However, PRRSV RNA is able to replicate in a variety of cell types with little species preference. Since the discovery of CD163 as an indispensable receptor, and MYH9 as an important factor for PRRSV, CD163s from various species have been shown to render nonpermissive cell lines susceptible to PRRSV, but this observation cannot explain why swine are still the sole host for PRRSV. How dose PRRSV hamper host development of an effective immune response? Swine infected by PRRSV (including most vaccine strains) develop delayed humoral and weak cell-mediated immune responses. Notably, upregulation of IL-10 and induction of regulatory T cells (Tregs) is a common feature that leaves hosts more susceptible to secondary infection. However, little is known about the mechanism behind PRRSV manipulation of the host immune system Do any conserved immune epitopes exist in PRRSV? Except for the identification of certain linear epitopes from PRRSV envelope proteins, which may not be well conserved among strains or genotypes, the protein structure of PRRSV envelope antigens is largely unknown. Although pig sera containing broad neutralizing antibodies against both genotypes have been reported, it is still uncertain if any conserved epitopes (preferably conformational epitopes) exist across PRRSV genotypes or strains. How could an effective vaccine against heterogeneous PRRSVs be developed? One reasonable explanation for the poor efficacy of available vaccines is the heterologous nature of different PRRSV isolates that results in drastic changes in antigenic determinants. Since certain advances have been made to develop a vaccine that confers heterologous protection, more investigation is needed to achieve complete protection of immunized swine from wild-type PRRSV challenge.

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Acknowledgments We thank Dr Chunyan Wu (Northwest A&F University) for critical review of the manuscript. Support for this work was received from the National Natural Science Foundation of China (31430084).

Supplemental Information Supplemental information associated with this article can be found [406_TD$IF]online at http://dx.doi.org/10.1016/j.tim.2017.06.001.

References 1.

Lunney, J.K. et al. (2016) Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune system. Annu. Rev. Anim. Biosci. 4, 129–154

19. Wang, D. et al. (2013) MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-kappaB pathway. PLoS One 8, e55838

2.

Mardassi, H. et al. (1994) Identification of major differences in the nucleocapsid protein genes of a Quebec strain and European strains of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 75, 681–685

20. Zhang, Q. et al. (2014) MicroRNA-23 inhibits PRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons. Virology 450–451 182–195

3.

Zhang, Q. and Yoo, D. (2015) PRRS virus receptors and their role for pathogenesis. Vet. Microbiol. 177, 229–241

4.

Butler, J.E. et al. (2014) Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic. Immunol. Res. 59, 81–108

5.

6.

7.

8.

9.

Balasuriya, U.B. and MacLachlan, N.J. (2004) The immune response to equine arteritis virus: potential lessons for other arteriviruses. Vet. Immunol. Immunopathol. 102, 107–129 Rowland, R.R. and Morrison, R.B. (2012) Challenges and opportunities for the control and elimination of porcine reproductive and respiratory syndrome virus. Transbound. Emerg. Dis. 59 (Suppl. 1), 55–59 Gao, J. et al. (2016) MYH9 is an essential factor for porcine reproductive and respiratory syndrome virus infection. Sci. Rep. 6, 25120 Burkard, C. et al. (2017) Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathog. 13, e1006206 Whitworth, K.M. et al. (2016) Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat. Biotechnol. 34, 20–22

10. Van Gorp, H. et al. (2010) Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. J. Virol. 84, 3101–3105 11. Chen, Y. et al. (2014) Additive inhibition of porcine reproductive and respiratory syndrome virus infection with the soluble sialoadhesin and CD163 receptors. Virus Res. 179, 85–92 12. Zhou, E.M. et al. (2008) Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus. J. Virol. Methods 149, 300–308 13. Yu, Y. et al. (2015) Single-chain anti-idiotypic antibody retains its specificity to porcine reproductive and respiratory syndrome virus GP5. Immunol. Lett. 163, 8–13 14. Straight, A.F. et al. (2003) Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 299, 1743– 1747 15. Liu, F. et al. (2017) New perspective of host microRNAs in the control of PRRSV infection. Vet. Microbiol. Published online January 9, 2017. http://dx.doi.org/10.1016/j.vetmic.2017.01. 004

21. Chen, J. et al. (2016) MicroRNA-373 facilitated the replication of porcine reproductive and respiratory syndrome virus by its negative regulation of type I interferon induction. J. Virol. 91, e01311–16 22. Zhou, M. et al. (2016) miRNA29 Promotes viral replication during early stage of PRRSV infection in vitro. DNA Cell Biol. 35, 636– 642 23. Zhang, Q. et al. (2016) MicroRNA-30c modulates type I IFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1. J. Immunol. 196, 2272–2282 24. Gao, L. et al. (2013) MicroRNA 181 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163. J. Virol. 87, 8808–8812 25. Li, N. et al. (2016) MicroRNA let-7f-5p Inhibits porcine reproductive and respiratory syndrome virus by targeting MYH9. Sci. Rep. 6, 34332 26. Wu, J. et al. (2014) MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151. Mol. Cell. Biochem. 394, 275–281 27. Zhang, A. et al. (2017) Heme oxygenase-1 metabolite biliverdin, not iron, inhibits porcine reproductive and respiratory syndrome virus replication. Free Radic. Biol. Med. 102, 149–161 28. Xiao, S. et al. (2015) MiR-24-3p promotes porcine reproductive and respiratory syndrome virus replication through suppression of heme oxygenase-1 expression. J. Virol. 89, 4527–4538 29. Xiao, S. et al. (2016) MiR-22 promotes porcine reproductive and respiratory syndrome virus replication by targeting the host factor HO-1. Vet. Microbiol. 192, 226–230 30. Shi, X. et al. (2015) Small interfering RNA targeting nonstructural protein1 alpha (nsp1alpha) of porcine reproductive and respiratory syndrome virus (PRRSV) can reduce the replication of PRRSV in MARC-145 cells. Res. Vet. Sci. 99, 215–217 31. Xie, J. et al. (2014) Inhibition of porcine reproductive and respiratory syndrome virus by specific siRNA targeting Nsp9 gene. Infect. Genet. Evol. 28, 64–70 32. Yang, M. et al. (2014) RNA interference targeting nucleocapsid protein inhibits porcine reproductive and respiratory syndrome virus replication in Marc-145 cells. J. Microbiol. 52, 333–339 33. Li, G. et al. (2007) Suppression of porcine reproductive and respiratory syndrome virus replication in MARC-145 cells by shRNA targeting ORF1 region. Virus Genes 35, 673–679 34. Li, L. et al. (2015) Cellular miR-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo. Sci. Rep. 5, 17010

16. Guo, X.K. et al. (2013) Increasing expression of microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection. J. Virol. 87, 1159–1171

35. Cao, S.F. et al. (2015) Inhibition of highly pathogenic porcine reproductive and respiratory syndrome virus replication by recombinant pseudorabies virus-mediated RNA interference in piglets. Vet. Microbiol. 181, 212–220

17. Li, L. et al. (2015) Host miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons. Virus Res. 195, 86–94

36. Li, L. et al. (2014) RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs. J. Biotechnol. 171, 17–24

18. Jia, X. et al. (2015) Cellular microRNA miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by activating innate antiviral immunity. Sci. Rep. 5, 10651

37. Han, X. et al. (2009) Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers. Antiviral Res. 82, 59–66

10

Trends in Microbiology, Month Year, Vol. xx, No. yy

TIMI 1473 No. of Pages 12

38. Opriessnig, T. et al. (2011) Inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptideconjugated morpholino oligomer. Antiviral Res. 91, 36–42

59. Bautista, E.M. and Molitor, T.W. (1999) IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages. Arch. Virol. 144, 1191–1200

39. Sun, N. et al. (2014) Sodium tanshinone IIA sulfonate inhibits porcine reproductive and respiratory syndrome virus via suppressing N gene expression and blocking virus-induced apoptosis. Antivir. Ther. 19, 89–95

60. Rowland, R.R. et al. (2001) Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine. Arch. Virol. 146, 539–555

40. Zhou, D.H. et al. (2013) Combination of low concentration of (–)-epigallocatechin gallate (EGCG) and curcumin strongly suppresses the growth of non-small cell lung cancer in vitro and in vivo through causing cell cycle arrest. Int. J. Mol. Sci. 14, 12023–12036 41. Zhao, C. et al. (2014) In vitro evaluation of the antiviral activity of the synthetic epigallocatechin gallate analog-epigallocatechin gallate (EGCG) palmitate against porcine reproductive and respiratory syndrome virus. Viruses 6, 938–950 42. Li, E. et al. (2015) In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus. Antivir. Ther. 20, 743–752 43. Cheng, J. et al. (2013) In vitro screening for compounds derived from traditional chinese medicines with antiviral activities against porcine reproductive and respiratory syndrome virus. J. Microbiol. Biotechnol. 23, 1076–1083 44. Yang, Q. et al. (2013) Inhibition of porcine reproductive and respiratory syndrome virus replication by flavaspidic acid AB. Antivir. Res. 97, 66–73 45. Ma, Z. et al. (2013) A novel compound from the mushroom Cryptoporus volvatus inhibits porcine reproductive and respiratory syndrome virus (PRRSV) in vitro. PLoS One 8, e79333 46. Zhang, S.L. et al. (2016) Anti-PRRSV effect and mechanism of tetrahydroaltersolanol C in vitro. J. Asian Nat. Prod. Res. 18, 303–314 47. Jiang, Y. et al. (2010) N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro. Vet. Res. Commun. 34, 607–617 48. Wang, Z.W. et al. (2013) In vitro antiviral activity and underlying molecular mechanisms of dipotassium glycyrrhetate against porcine reproductive and respiratory syndrome virus. Antivir. Ther. 18, 997–1004 49. Liu, H. et al. (2015) An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication. Vet. Microbiol. 181, 252– 260 50. Liu, H. et al. (2016) Intracellularly expressed nanobodies against non-structural protein 4 of porcine reproductive and respiratory syndrome virus inhibit virus replication. Biotechnol. Lett. 38, 1081–1088 51. Sang, Y. et al. (2008) Toll-like receptor 3 activation decreases porcine arterivirus infection. Viral Immunol. 21, 303–313 52. Miller, L.C. et al. (2009) Role of Toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus. Clin. Vaccine Immunol. 16, 360–365 53. Zhang, L. et al. (2013) Poly(I:C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3. Antivir. Res. 99, 197–206 54. Du, Y. et al. (2016) Synthetic Toll-like receptor 7 ligand inhibits porcine reproductive and respiratory syndrome virus infection in primary porcine alveolar macrophages. Antivir. Res. 131, 9–18 55. Zhang, L. et al. (2013) Toll-like receptor ligands enhance the protective effects of vaccination against porcine reproductive and respiratory syndrome virus in swine. Vet. Microbiol. 164, 253–260 56. Hu, Y. et al. (2016) Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-kappaB signaling pathway. Sci. Rep. 6, 23977 57. Nan, Y. et al. (2014) Interferon induction by RNA viruses and antagonism by viral pathogens. Viruses 6, 4999–5027 58. Overend, C. et al. (2007) Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 88, 925–931

61. Wang, R. et al. (2013) Variable interference with interferon signal transduction by different strains of porcine reproductive and respiratory syndrome virus. Vet. Microbiol. 166, 493–503 62. Dong, S. et al. (2012) Inhibitory effects of recombinant porcine interferon-alpha on high- and low-virulence porcine reproductive and respiratory syndrome viruses. Res. Vet. Sci. 93, 1060– 1065 63. Luo, R. et al. (2011) Antiviral activity of type I and type III interferons against porcine reproductive and respiratory syndrome virus (PRRSV). Antivir. Res. 91, 99–101 64. Carter, Q.L. and Curiel, R.E. (2005) Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection. Vet. Immunol. Immunopathol. 107, 105–118 65. Wee, G.J. et al. (2001) Efficacy of porcine reproductive and respiratory syndrome virus vaccine and porcine interleukin12. Vet. Ther. 2, 112–119 66. Meier, W.A. et al. (2004) Cytokines and synthetic doublestranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopathol. 102, 299–314 67. Xue, Q. et al. (2004) Immune responses of swine following DNA immunization with plasmids encoding porcine reproductive and respiratory syndrome virus ORFs 5 and 7, and porcine IL-2 and IFNgamma. Vet. Immunol. Immunopathol. 102, 291–298 68. Rompato, G. et al. (2006) Positive inductive effect of IL-2 on virus-specific cellular responses elicited by a PRRSV-ORF7 DNA vaccine in swine. Vet. Immunol. Immunopathol. 109, 151–160 69. Du, Y. et al. (2014) Highly efficient expression of interleukin-2 under the control of rabbit beta-globin intron II gene enhances protective immune responses of porcine reproductive and respiratory syndrome (PRRS) DNA vaccine in pigs. PLoS One 9, e90326 70. Charerntantanakul, W. and Kasinrerk, W. (2012) Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopathol. 146, 159–168 71. Tian, D. et al. (2017) Enhancing heterologous protection in pigs vaccinated with chimeric porcine reproductive and respiratory syndrome virus containing the full-length sequences of shuffled structural genes of multiple heterologous strains. Vaccine 35, 2427–2434 72. Sun, H. et al. (2016) Identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain. Virology 499, 313–321 73. Wang, R. et al. (2013) Enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain. Vaccine 31, 5537–5543 74. Fontanella, E. et al. (2017) An interferon inducing porcine reproductive and respiratory syndrome virus vaccine candidate elicits protection against challenge with the heterologous virulent type 2 strain VR-2385 in pigs. Vaccine 35, 125–131 75. Robinson, S.R. et al. (2015) Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus. Virus Res. 203, 56–65 76. Zhang, Y. et al. (1998) Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus. Vet. Microbiol. 63, 125–136 77. Ostrowski, M. et al. (2002) Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J. Virol. 76, 4241– 4250 78. Fan, B. et al. (2015) The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus

Trends in Microbiology, Month Year, Vol. xx, No. yy

11

TIMI 1473 No. of Pages 12

regulate viral neutralization susceptibility to the porcine serum neutralizing antibody. Virus Res. 204, 21–30 79. Kim, W.I. et al. (2013) Significance of genetic variation of PRRSV ORF5 in virus neutralization and molecular determinants corresponding to cross neutralization among PRRS viruses. Vet. Microbiol. 162, 10–22 80. Van Breedam, W. et al. (2011) Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus. Vet. Immunol. Immunopathol. 141, 246–257 81. Li, J. and Murtaugh, M.P. (2012) Dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes. Virology 433, 367–376 82. Vanhee, M. et al. (2011) Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies. Vaccine 29, 4794– 4804 83. Weiland, E. et al. (1999) Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet. Microbiol. 66, 171–186 84. Costers, S. et al. (2010) GP4-specific neutralizing antibodies might be a driving force in PRRSV evolution. Virus Res. 154, 104–113 85. Vanhee, M. et al. (2010) A variable region in GP4 of Europeantype porcine reproductive and respiratory syndrome virus induces neutralizing antibodies against homologous but not heterologous virus strains. Viral Immunol. 23, 403–413 86. Trible, B.R. et al. (2015) A single amino acid deletion in the matrix protein of porcine reproductive and respiratory syndrome virus confers resistance to a polyclonal swine antibody with broadly neutralizing activity. J. Virol. 89, 6515–6520 87. Leng, C.L. et al. (2012) Highly pathogenic porcine reproductive and respiratory syndrome virus GP5B antigenic region is not a neutralizing antigenic region. Vet. Microbiol. 159, 273–281 88. Zhou, L. et al. (2013) Broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the GP4 or M genes. PLoS One 8, e66645 89. Vu, H.L. et al. (2015) A synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection. J. Virol. 89, 12070–12083 90. Kim, H. et al. (2011) The assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods. Virol. J. 8, 323 91. Papatsiros, V.G. et al. (2006) Long-term administration of a commercial porcine reproductive and respiratory syndrome virus (PRRSV)-inactivated vaccine in PRRSV-endemically infected sows. J. Vet. Med. B Infect. Dis. Vet. Pub. Health 53, 266–272 92. Doria-Rose, N.A. et al. (2009) Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies. J. Virol. 83, 188–199 93. Rowland, R.R. et al. (2003) Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero. Vet. Microbiol. 96, 219–235 94. Mengeling, W.L. et al. (1998) Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of ‘atypical' PRRS. Am. J. Vet. Res. 59, 1540–1544 95. Han, J. et al. (2006) Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus. Virus Res. 122, 175–182 96. Tian, K. et al. (2007) Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS One 2, e526

12

Trends in Microbiology, Month Year, Vol. xx, No. yy

97. An, T.Q. et al. (2011) Highly pathogenic porcine reproductive and respiratory syndrome virus, Asia. Emerg. Infect. Dis. 17, 1782–1784 98. Rajkhowa, T.K. et al. (2015) Porcine reproductive and respiratory syndrome virus (PRRSV) from the first outbreak of India shows close relationship with the highly pathogenic variant of China. Vet. Q. 35, 186–193 99. Zhou, L. et al. (2015) NADC30-like strain of porcine reproductive and respiratory syndrome virus, China. Emerg. Infect. Dis. 21, 2256–2257 100. Wang, C. et al. (2010) Phylogenetic analysis and molecular characteristics of seven variant Chinese field isolates of PRRSV. BMC Microbiol. 10, 146 101. Madsen, K.G. et al. (1998) Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds. Arch. Virol. 143, 1683–1700 102. Botner, A. et al. (1997) Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine. Vet. Rec. 141, 497–499 103. Wenhui, L. et al. (2012) Complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (PRRSV) strain: evidence for recombination between vaccine and wild-type PRRSV strains. J. Virol. 86, 9543 104. Charerntantanakul, W. (2012) Porcine reproductive and respiratory syndrome virus vaccines: Immunogenicity, efficacy and safety aspects. World J. Virol. 1, 23–30 105. Opriessnig, T. et al. (2002) Comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (ingelvac PRRS MLV), the parent strain of the vaccine (ATCC VR2332), ATCC VR2385, and two recent field isolates of PRRSV. J. Virol. 76, 11837–11844 106. Patel, D. et al. (2010) Porcine reproductive and respiratory syndrome virus inhibits type I interferon signaling by blocking STAT1/STAT2 nuclear translocation. J. Virol. 84, 11045–11055 107. Wang, R. and Zhang, Y.J. (2014) Antagonizing interferon-mediated immune response by porcine reproductive and respiratory syndrome virus. BioMed. Res. Int. 2014, 315470 108. Yang, L. et al. (2017) Porcine reproductive and respiratory syndrome virus antagonizes JAK/STAT3 signaling via nsp5, which induces STAT3 degradation. J. Virol. 91, e02087-16 109. Chen, Z. et al. (2010) Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist. Virology 398, 87–97 110. Li, H. et al. (2010) The cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein 2 antagonizes interferon regulatory factor 3 activation. J. Gen. Virol. 91, 2947–2958 111. Sun, Z. et al. (2010) The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions. J. Virol. 84, 7832–7846 112. Huang, C. et al. (2014) Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-kappaB essential modulator. J. Virol. 88, 10934–10945 113. Shi, X. et al. (2011) Endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit IFN-beta induction. Mol. Immunol. 48, 1568– 1572 114. Sagong, M. and Lee, C. (2011) Porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting IRF3 activation in immortalized porcine alveolar macrophages. Arch. Virol. 156, 2187–2195 115. Wang, R. et al. (2013) Porcine reproductive and respiratory syndrome virus Nsp1beta inhibits interferon-activated JAK/ STAT signal transduction by inducing karyopherin-alpha1 degradation. J. Virol. 87, 5219–5228