ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) is a target of diversifying selection as infection progresses from acute infection to virus rebound

Infection, Genetics and Evolution 40 (2016) 167–175

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Research paper

ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) is a target of diversifying selection as infection progresses from acute infection to virus rebound Nanhua Chen a,b,⁎, Benjamin R. Trible a, Maureen A. Kerrigan a, Kegong Tian c, Raymond R.R. Rowland a a b c

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China OIE Porcine Reproductive and Respiratory Syndrome Reference Laboratory, Beijing, PR China

a r t i c l e

i n f o

Article history: Received 30 January 2016 Received in revised form 28 February 2016 Accepted 2 March 2016 Available online 4 March 2016 Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Viral quasispecies Virus rebound Deep sequencing Diversifying selection

a b s t r a c t Genetic variation in both structural and nonstructural genes is a key factor in the capacity of porcine reproductive and respiratory syndrome virus (PRRSV) to evade host defenses and maintain within animals, farms and metapopulations. However, the exact mechanisms by which genetic variation contribute to immune evasion remain unclear. In a study to understand the role of host genetics in disease resistance, a population of pigs were experimentally infected with a type 2 PRRSV isolate. Four pigs that showed virus rebound at 42 days post-infection (dpi) were analyzed by 454 sequencing to characterize the rebound quasispecies. Deep sequencing of variable regions in nsp1, nsp2, ORF3 and ORF5 showed the largest number of nucleotide substitutions at day 28 compared to days 4 and 42 post-infection. Differences were also found in genetic variations when comparing tonsil versus serum. The results of dN/dS ratios showed that the same regions evolved under negative selection. However, eight amino acid sites were identified as possessing significant levels of positive selection, including A27V and N32S substitutions in the GP5 ectodomain region. These changes may alter GP5 peptide signal sequence processing and N-glycosylation, respectively. The results indicate that the greatest genetic diversity occurs during the transition between acute and rebound stages of infection, and the introduction of mutations that may result in a gain of fitness provides a potential mechanism for persistence. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease of pigs, worldwide. The estimated cost to US producers alone is $664 million per year (Holtkamp et al., 2013). The etiologic agent, PRRS virus (PRRSV), is an enveloped, positive-sense, single-stranded RNA virus belonging to the genus Arterivirus, family Arteriviridae, order Nidovirales (Cavanagh, 1997). The principal clinical signs associated with PRRSV infection are lateterm reproductive disorders in pregnant gilts and sows, respiratory problems in young piglets, and poor growth performance in growing pigs (Wensvoort et al., 1991). PRRSV is easily transmitted vertically and horizontally, has the capacity to remain as a subclinical infection for a relatively prolonged time, and participates as a cofactor in several polymicrobial syndromes, such as porcine respiratory disease complex (PRDC) and porcine circovirus-associated disease (PCVAD) (Gillespie et al., 2009; Thacker, 2001).

⁎ Corresponding author at: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, PR China. E-mail address: [email protected] (N. Chen).

http://dx.doi.org/10.1016/j.meegid.2016.03.002 1567-1348/© 2016 Elsevier B.V. All rights reserved.

The PRRSV genome is about 15 kb, possessing a 5′-capped positive RNA genome, a 3′-polyadenylated tail, and ten open reading frames (ORFs) flanked by 5′- and 3′- untranslated regions (UTR) (Lunney et al., 2016). ORF1a and ORF1b encode two nonstructural polyproteins, pp1a and pp1ab, which are proteolytically cleaved to yield at least 16 nonstructural proteins (nsp1α, nsp1β, nsp2, nsp2N, nsp2TF, nsp3–6, nsp7α, nsp7β, nsp8–12) (Fang and Snijder, 2010; Fang et al., 2012). ORF2 through ORF7 encode eight structural proteins (GP2a, E (2b), GP3, GP4, 5a, GP5, M, N) (Firth et al., 2011; Johnson et al., 2011; Music and Gagnon, 2010). GP5 and M, which form a heterodimer, are the predominant viral surface proteins (Mardassi et al., 1996). The minor surface glycoproteins, GP2a, GP3 and GP4, form a heterotrimer, and presumably interact with the CD163 receptor (Wissink et al., 2005), which is essential for PRRSV infection (Whitworth et al., 2016). The small protein, E, is likely to form a pore complex involved in virion uncoating in the cytoplasm (Lee and Yoo, 2006). Another recently identified small protein, 5a, is essential for virus viability (Firth et al., 2011; Johnson et al., 2011; Sun et al., 2013). The sole component of the PRRSV capsid, N, also plays a nonstructural role in the nucleus/nucleolus (Rowland et al., 1999a). All nonstructural and structural proteins are required for virus replication (Music and Gagnon, 2010; Welch et al., 2004).

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In nature, PRRSV exists as a swarm of diverse variants known as a quasispecies (Goldberg et al., 2003; Rowland et al., 1999b). It is one of the most rapidly evolving viruses known, with an estimated evolutionary rate of 4.7–9.8 × 10− 2/site/year (Hanada et al., 2005), producing a high degree of genetic variation among PRRSV isolates as well as sequence variants within an infected pig (Chang et al., 2002; Meng et al., 1995a; Shi et al., 2010a,b; Stadejek et al., 2008). PRRSV has two genotypes (Meng et al., 1995a; Nelsen et al., 1999), Type 1 and Type 2. Type 1 PRRSV can be divided into three subtypes with subtype 1 being further classified into 12 clades with inter-clade genetic distances of N10% (Shi et al., 2010a; Stadejek et al., 2008). Type 2 PRRSV can be divided into 9 lineages with at least 11% inter-lineage diversity (Shi et al., 2010b). Chang et al. (2002) observed the appearance of amino acid (aa) variants during sequential passages in pigs and detected multiple variants in ORF5 in a single pig. The roles that genetic variability play in PRRSV persistence remain unclear, but are likely related to evasion of innate and adaptive immune responses (Chand et al., 2012). Nucleotide variation is not evenly distributed across the PRRSV genome, but is concentrated in “hotspots” possessing sequence variability and hypervariability, which are located in both nonstructural and structural regions on the genome. For example, nsp1 and nsp2 are more variable than other nonstructural regions (Darwich et al., 2011; Fang et al., 2004) and structural genes ORF3 and ORF5 each contain an important variable domain (Mardassi et al., 1995; Meng et al., 1995b). Presumably, mutations in variable regions play crucial roles in escape from host defenses (Ansari et al., 2006; Beura et al., 2010; Darwich et al., 2011; Goldberg et al., 2003; Rowland et al., 1999b; Sun et al., 2010; Sun et al., 2012; Vu et al., 2011). The best-studied example is the ectodomain region of the ORF5 encoded protein, GP5, which possesses a highly conserved domain flanked by conserved glycosylation sites and hypervariable domains. The distal hypervariable region possesses N-glycosylation sites, the number and location of which can change over the course of infection. Presumably, enhanced glycosylation combined with peptide sequence hypervariability protects a conserved epitope from neutralizing antibody (nAb): an immunological escape strategy described for gp120 of HIV (Wei et al., 2003). During PRRSV infection, viremia typically reaches a peak between 7 and 21 days post-infection (dpi) followed by the decay and eventual disappearance of detectable virus from the blood at about 35 to 42 dpi (Lopez and Osorio, 2004). Low levels of virus remain in lymphoid tissues for an extended period of time (Allende et al., 2000; Rowland et al., 2003). However, after achieving an initial peak, virus levels in the blood can show secondary peaks, which are termed “virus rebound” (Reiner et al., 2010). In a study directed at identifying genomic markers linked to PRRS through the experimental infection of several hundred pigs, subpopulations that experienced virus rebound were identified (Boddicker et al., 2012; Rowland et al., 2012). Rebound virus may represent a variant virus within the quasispecies that attains a new adaptive peak, either through a change in tropism or by escape from adaptive immunity. In a previous study, we examined PRRSV evolution in pigs that lack B and T cells, a condition referred to as severe combined immunodeficiency (SCID) (Chen et al., 2015). Viremia was lower in SCID pigs than in normal littermates at 4 dpi but was significantly elevated by 21 dpi. Only seven aa substitutions were identified over the course of acute infection, all of which occurred in both SCID and normal pigs and appeared very early after infection. The conclusion was that these aa substitutions were not related to adaptive immune selection, but likely the process of a tissue culture-adapted virus becoming re-adapted to its natural host. The purpose of this study was to analyze genetic variation during both acute and rebound phases of infection. The pigs in this study were from a population of 200 experimentally infected pigs and were selected from a subpopulation that showed virus rebound in the blood. The areas of the genome targeted for sequencing were previously

identified as hypervariable located in the non-structural domains nsp1 and nsp2, and the structural genes ORF3 and ORF5 (Brar et al., 2014; Darwich et al., 2011; Fang et al., 2004; Mardassi et al., 1995; Meng et al., 1995b). The approach used 454 sequencing to cover each hypervariable region in a single ~ 400 bp sequencing reaction. The results showed large variations between animals in terms of the number and location of nucleotide substitutions. However, all pigs showed the greatest variation at 28 dpi and all had aa substitutions in the ectodomain region of GP5. 2. Materials and methods 2.1. Virus and pigs All studies involving animals and viruses were performed after approval by the Kansas State University institutional biosafety and animal care and use committees. The experimental design included the infection of 200 three-week-old pigs with a Type 2 PRRSV isolate, NVSL 97-7895 (GenBank accession number AY545985). Blood samples were collected at 0, 4, 7, 11, 14, 21, 28, 35, and 42 dpi and serum was stored at −80 °C. PRRSV viremia was measured using a AgPath-ID™ NA & EU PRRSV One-Step Multiple qRT-PCR Kit (Applied Biosystems) according to the manufacturer's recommendations. The results were reported as the log10 copies per 25 μl reaction. 2.2. Measurement of virus-neutralizing activity Virus neutralization (VN) activity was measured in serum as previously described (Trible et al., 2015). Briefly, MARC-145 cells were propagated and maintained in Minimum Essential Media (MEM, Fisher Scientific) containing 7% fetal bovine serum (FBS, Gibco), 80 U/ml penicillin–streptomycin (Gibco) and 0.3 μg/ml Fungizone (Gibco) at 37 °C with 5% CO2. Two-fold serial dilutions of sera in 100 μl of media, beginning at a dilution of 1:8, were added to 100 μl containing 200 TCID50 of virus and incubated for 1 h at 37 °C and then added to confluent MARC-145 cells in a 96 well plate. After 4 days, the monolayers were examined for PRRSV-induced cytopathic effect (CPE). The VN titer was reported as the reciprocal of the highest dilution in which no CPE was observed. 2.3. DNA library preparation and 454-pyrosequencing Total RNA was extracted from 100 μl of tissue culture medium or serum, or 50 mg of tonsil. Samples were homogenized in 1 ml TRIzol® Reagent (Invitrogen) according to the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987) and eluted in 50 μl RNase-free water. cDNA was generated by reverse transcription using random hexamer primers and the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Preparation of the amplicon library involved two rounds of PCR (Daigle et al., 2011). The first round was performed using virus sequence-specific primers, listed in Table S1. The variable and hypervariable regions were selected based on the comparison of 20 genomic sequences from GenBank. The regions selected for study are shown in Fig. S1. A second round of amplification incorporated 454-adaptor multiplex identifier (MID) primers (Table S2). The primers were designed to yield products between 447 bp and 542 bp in length. For unidirectional sequencing, MIDs were included only on forward primers for the second round of PCR. A different MID was used for each of the 15 samples with the same MID being used for all 9 amplicons of the hypervariable regions of PRRSV from a given sample. The annealing temperature was set at 60 °C for both rounds of PCR. The final concentrations of primers and DNA polymerase were 0.3 μM and 0.02 U/μl, respectively. The other reaction components and thermocycling conditions were set according to manufacturer's

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recommendations for the Phusion high fidelity DNA polymerase (New England Biolabs). After the second round of amplification, the amplicons were purified with Agencourt AMPure XP 5 ml Kit (Beckman Coulter). The concentration of each amplicon was measured using a NanoDrop ND8000 Spectrophotometer (Thermo Scientific). Values were converted to molecules/μl using the formula: molecules/μl = [sample conc. (ng/μl) × 6.022 × 1023] / [656 × 109 × amplicon length (bp)] Each amplicon concentration was normalized by dilution and equal volumes of the normalized amplicons were combined to create a library containing 135 amplicons. The amplicon library was diluted in 1× TE buffer to a final concentration of 1 × 107 molecules/μl for each amplicon and stored at −20 °C. Emulsion PCR (emPCR) amplification and 454 deep sequencing were performed on the amplicon library by the DNA sequencing facility in the Department of Plant Pathology, Kansas State University. The Lib-L emPCR Kit (Roche) was used for amplification according to the manufacturer's directions. The GS FLX Titanium Sequencing Kit XLR70 (Roche) was used for 454 sequencing according to the manufacturer's instructions. Sequencing was performed on a GS FLX + Instrument using the 454 Sequencing System Software Version 2.6 package for analysis. Reads for each sample were sorted according to the MID. Sequence reads were mapped against the reference sequence from the parental virus (AY545985) using 454 Life Sciences GS Reference Mapper (Version 2.6). The minimum overlap length was 40 and the minimum overlap identity was 90%. Coverage (number of reads per amplicon) was calculated and variants were called. Variants were further filtered based on the coverage, variant frequency, and homopolymer as described previously (Chen et al., 2015). Only high-confidence singlenucleotide variants with the following features were selected: 1) at least 3 non-duplicate reads contained the nucleotide substitution; 2) a substitution frequency higher than 5%; and 3) not located at a homopolymer site. All mutations were further confirmed by the sequencing assembly visualization software, Tablet (Milne et al., 2013). 2.4. Amino acid substitution rates and selection All nucleotide substitutions in the nine amplicon regions of a given sample were assembled into one sequence for the analysis of selective pressures on the substitutions. Each sample's representative sequence was compared to the representative sequence assembled from the virus used for infection. The ratios of non-synonymous (dN) and synonymous (dS) substitutions for codon-aligned sequences from each sample were calculated using the Synonymous Non-synonymous Analysis Program (SNAP v2.1.1) (http://www.hiv.lanl.gov/content/

169

sequence/SNAP/SNAP.html) based on the method of Nei and Gojobori (Nei and Gojobori, 1986) to yield the equation Ω = dN/dS. Values of Ω = 1, Ω b 1 or Ω N 1 indicate that the protein-coding gene is under neutral, negative, or positive selection, respectively (Yang and Bielawski, 2000). One-way analysis of variation followed by Dunn's multiple comparison test was used to evaluate differences between mean Ω at 4, 28, and 42 dpi using Graphpad Prism version 6.07. Positive selection pressure for each aa position was calculated using the CodeML program in PAMLX software (Xu and Yang, 2013). The files (in the format of phylip and trees) of codon-aligned sequences from all samples were input into CodeML program and tested using the codon substitution models M0, M1, M2, M7 and M8 (Yang and Bielawski, 2000). The existence and degree of positive selection was measured using likelihood ratio tests (LRT) by two paired comparisons of the model M2 (selection) with the model M1 (neutral) and the model M8 (beta & Ω) with the model M7 (beta). The LRT statistic or twice the log likelihood differences between the two model pairs (2Δl) were compared using a Chi-square distribution with critical value of 9.21 (degrees of freedom (df) = 2) at a 1% significance level (Delisle et al., 2012; Yang, 2007). Afterwards, the positively selected sites were calculated and the Bayes Empirical Bayes (BEB) method was used to calculate the posterior probability that a codon belonged to the class of Ω N 1, so inferring positively selected codons at 95% and 99% significance (Yang et al., 2005).

3. Results 3.1. PRRSV infection shows rebound viremia The viremia results for 141 experimentally infected pigs having sera collected at all time points are shown in Fig. 1A. Real-Time PCR results confirmed that all pigs were productively infected. Viremia peaked between 4 and 14 dpi with values ranging between 5.5 and 7.5 log10 templates/rxn. Viremia levels decayed until virus was no longer detected in serum of most pigs. Approximately 30% or 43 of the 141 pigs showed subsequent peaks in viremia indicating virus rebound. Rebound followed several patterns over the 42-day infection period, including pigs with two rebound viremia peaks. Four pigs with the highest viremia levels at 42 dpi were selected for further study (see Fig. 1B). All four pigs showed similar viremia patterns that included an initial peak followed by a period of decay and two subsequent rebound peaks. The first rebound peak in these pigs occurred at 28 dpi and virus loads were significantly lower (4.39 ± 1.06) than the initial peak at 4 dpi (5.77 ± 0.81) (p = 0.04). Virus loads in the second rebound peak at 42 dpi (4.95 ± 0.58) were not significantly different from either 4 dpi or 28 dpi. For the purpose of comparison, the solid line shows the mean viremia for the 141 pigs.

Fig. 1. Viremia in pigs infected with PRRSV. (A) Viremia of 141 pigs over 42 dpi. (B) Four pigs with rebound viremia were selected for deep sequencing.

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3.2. Quasispecies analysis of the nine amplicon regions

Table 1 Virus neutralizing activity against NVSL 97-7895. Virus neutralization titer Pig 6576 6685 6721 6774

Day 4

Day 28

Day 42

b8⁎ b8 b8 b8

16 b8 b8 b8

b8 32 8 16

⁎ Neutralization titer showing inverse of the highest serum dilution with VN activity. Titer b 8 indicates no detectable virus neutralization activity.

In our previous work, differing degrees of neutralizing activity (NA) in serum following PRRSV infection were identified; ranging from only homologous NA (neutralization of only the virus used for infection) to heterologous/broad NA (the capacity to neutralize several PRRSV isolates) (Trible et al., 2015). In this study, NA was measured in serum at 4, 28, and 42 dpi against the virus used for infection, NVSL 97-7895 (NVSL), and two heterologous viruses, VR-2332 and KS06, which were 91% and 89% identical to NVSL at the peptide sequence level, respectively. As shown in Table 1, these four pigs showed detectable NA against NVSL, the homologous virus, at some time point during the study period. NA was not detected when KS06 and VR-2332 were used as targets for neutralization (data not shown). Thus, these pigs displayed primarily homologous NA.

In order to have sufficient quantities of virus for 454 sequencing over the 42-day infection period, we selected pigs that possessed relatively high levels of viremia at both initial and subsequent peaks (4, 28 and 42 dpi). Both rounds of PCR yielded nine products of the expected sizes for all four pigs at all time points. A total of 135 amplicons were sequenced including samples from serum (27 amplicons per pig), tonsil (18 amplicons) and the inoculum (9 amplicons) used for infection. Coverage ranged between 118 and 7167 reads per amplicon with an average of 872. The total number of nucleotide substitutions covering the 3113 nucleotide 9 amplicon region for the four pigs is summarized in Fig. 2. The results showed that the number of substitutions varied between pigs and days after infection, but all pigs showed the greatest number at 28 dpi. Over the nine amplicon segments, three of the four pigs showed a greater than six-fold increase in the number of nucleotide substitutions on day 28 compared to day 4. Results for dN, dS and Ω are shown in Table 2. The Ω value for one serum sample, pig 6774 at 42 dpi, was 1.566, suggesting that the virus in pig 6774 at 42 dpi was under positive selection. However, for all other samples, Ω was b1 indicating negative selection. Compared to 4 dpi (mean Ω = 0.6933 ± 0.1248), the mean Ω value for 28 dpi (0.2824 ± 0.0988) was significantly lower (p = 0.02), indicating greater negative selection pressure at 28 dpi than 4 dpi. At 42 dpi, the Ω values (0.7573 ± 0.5415) varied widely between pigs.

Fig. 2. Locations and frequencies of nucleotide substitutions. Substitutions at 4 dpi, 28 dpi and 42 dpi in sera from four pigs. The amplified regions of PRRSV genome were highlighted with solid lines.

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Table 2 Rates for nonsynonymous and synonymous substitutions.1 Day 4 Pig dN dS Ω (dN/dS) Mean Ω ± SD 1 2

6576 6685 0.0021 0.0029 0.0040 0.0040 0.5250 0.7250 0.6933 ± 0.1248a2

Day 28 6721 0.0033 0.0040 0.8250

6774 0.0037 0.0053 0.6981

Day 42

6576 6685 0.0108 0.0100 0.0698 0.0257 0.1547 0.3891 0.2824 ± 0.0988b

6721 0.0104 0.0326 0.3190

6774 0.0209 0.0783 0.2669

6576 0.0058 0.0121 0.4793

6685 6721 0.0104 0.0087 0.0188 0.0202 0.5532 0.4307 0.7573 ± 0.5415ab

6774 0.0083 0.0053 1.5660

Substitution rates for nonsynonymous and synonymous sites within the 9 amplicon region were calculated using SNAP (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html). Means with different letters are significantly different (p b 0.05).

Fig. 3 shows the number of nucleotide substitutions in each amplified segment. The number of substitutions within the 9 amplicon regions differed between animals and time points. For example, at 28 dpi pigs 6576, 6721, 6774 each had about 20 substitutions in amplicon 1 in the nsp1 region, but pig 6685 did not have any substitutions in amplicon 1. A similar pattern was seen for amplicon 7, located near the 3′ end of nsp2 (see Fig. 3). There were no significant differences in the number of substitutions for amplicon 8, which covered a hypervariable region in ORF3. For pig 6774, an increased number of substitutions at day 28 were found in most amplicon regions, except for amplicons 4, 6 and 8. Even though there were differences within a pig and between pigs, all pigs shared a day 28 increase in amplicon 9. Amplicon 9 covered the ORF5 region, which codes for the major surface glycoprotein and forms a disulfide bond with the conserved matrix protein. A detailed illustration showing the ectodomain region of GP5 is shown in Fig. 4. No aa substitutions were seen in the ectodomain region at 4 dpi. By day 28, 14 unique aa substitutions were found, with most of the changes appearing within the HV-1 domain. Thaa et al. (Thaa et al., 2013) first described that a signal peptide sequence cleavage site is located between aa 31 and 32 with an alternative cleavage site located upstream between aa 26 and 27. Cleavage at the 31/32 site results in the removal of a decoy epitope known as epitope A (Ostrowski et al., 2002). Cleavage at the 26/27 site retains epitope A. Several of the substitutions were located in the vicinity of the 26/ 27 and 31/32 cleavage sites. The same aa substitutions, 27-ALV to 27-VLG, were found in all four pigs. At 42 dpi the 27-V change was retained in three pigs, while pig 6685, had an A to V change at position 26; the G at position 29 reverted to V by 42 dpi in all four pigs. The 4-K, 16-F, and 51-R changes were also found in all four pigs but only at 28 dpi, indicating they are transient deleterious mutations that are later purged by negative selection. The three ectodomain regions containing the 27-ALV, 27-VLG, and 26-VAL aa sequences were analyzed for the location of predicted cleavage sites using the SignalP 4.0 webbased program (Petersen et al., 2011). The 27-ALV sequence, found in the inoculum virus and at 4 dpi, had two predicated cleavage sites, located between aa 26/27 and 31/32. Even though the C-scores are similar, experimental evidence indicates that the preferred signal peptide cleavage site is between aa 31/32. The outcome is the removal of the epitope A and loss of the decoy epitope. Substituting 27-VLG for 27-ALV results in a reduction in the C-score for the 31/32 site and favoring the upstream 26/27 site. The net effect would be the retention of the epitope A decoy epitope as well as the preservation of a new glycosylation site resulting from a N to S substitution at position 32 (see pig 6685 at 28 dpi, and pigs 6721 and 6774 at 42 dpi). The A-V substitution at position 27 results in reduced C-scores for both cleavage sites (Fig. 5). Eight aa sites located within the 9-amplicon regions were shown to evolve under positive selection (Table 3). Selection pressures were calculated using models M2 (2Δl = 50.7) and M8 (2Δl = 50.5) with a Chi-square critical value of 9.21 (df = 2) for a 1% significance level. These positively selected sites are spread in all amplified regions, which included the A27V and the N32S substitutions in GP5 locating at the edge of signal peptide cleavage sites (Thaa et al., 2013). The A27V substitution resided at the beginning of the decoy epitope A and the N32S substitution was right after the decoy epitope and created a new

N-linked glycosylation site (from NAN to NAS) (Fig. 4). The frequencies of some of the eight mutations decreased during infection (Table 3). For instance, the cytosine to thymine substitution at position 287 in ORF3 (P96L in GP3) was 44% and 43% in the inoculum and 4 dpi sera, respectively, but decreased to 6% in the 42 dpi sera, indicating that it's the 287 position evolved under positive selection rather than the thymine mutation were positively selected. The reverse mutation was positively

Fig. 3. The number of nucleotide substitutions in each amplicon. The number of substitutions at 4 dpi, 28 dpi and 42 dpi was shown in white, gray and black bars, respectively.

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Fig. 4. Amino acid substitutions in the ectodomain region of GP5. The alignment of amino acid sequences of GP5 covering the 60 amino acids in the ectodomain region. The peptide signal sequence is marked by amino acids in bold italics. An alternative peptide signal sequence cleavage site is shown by the arrow (Thaa et al., 2013). Asterisks identify the location of the two conserved glycosylation sites. Between the two glycosylation sites is a cysteine shown by a triangle, which forms a disulfide bond with the matrix protein. Flanking the two glycosylation sites are two hypervariable domains, HV-1 and HV-2. Other predicted glycosylation sites are shown as gray boxes. The decoy epitope (epitope A) and neutralizing epitope (epitope B) are underlined (Ostrowski et al., 2002).

selected in this case. The mutations in GP5 did not result in any aa changes in 5a (data not shown), which is consistent with the hypothesis that selective codon usage driven by purifying selection maintains the conservation of 5a RQ-rich motif (Robinson et al., 2013). 3.3. Comparison of tonsils and sera collected at 42 dpi from the same pigs The tonsil is generally considered a site of persistent replication (Rowland et al., 2003; Wills et al., 1997). A comparison of the nucleotide substitutions between serum and tonsil is shown in Table 4. Three substitutions were found in serum but not tonsil and five were found in tonsil but not serum. The eight discrepant substitutions were present at relatively high frequencies, ranging between 10 and 34%. Additionally, 16 nucleotide substitutions were found in both serum and tonsil samples, however, the frequencies varied widely. For instance, the C80T substitution in ORF5 was found in 70% of the serum replicons from pig 6576, but 35% of the tonsil replicons from the same pig. The C230T replacement in nsp1α was found in 95% of the serum replicons from pig 6721, but 24% of the tonsil replicons. The results show that genetic variations are different in serum and tonsil, suggesting that selection pressures differ between tissues, even within the same animal. 4. Discussion Due to its high mutation rate and lack of proofreading-repair ability, PRRSV is a rapidly evolving virus (Hanada et al., 2005; Steinhauer et al., 1992). As a consequence, PRRSV circulates in vivo as a quasispecies with a dynamic distribution that is subjected to a continuous process of

genetic variation, competition and selection (Farci, 2011). The genomic heterogeneity allows the virus quasispecies to rapidly adapt to changes in the microenvironment such as the progression of the host immune response (Lauring and Andino, 2010; Lopez-Bueno et al., 2003). These factors likely play a role in virus rebound in pigs (Fig. 1). Deep sequencing with 454-pyrosequencing technology is a useful platform for the study of genetic diversity (Astrovskaya et al., 2011; Brar et al., 2014; Chen et al., 2015). The high throughput capacity enables deep coverage of each variable region facilitating the detection of mutations even at low frequencies (Astrovskaya et al., 2011). Using deep sequencing, this study identified substitutions (based on at least hundreds of reads for each amplicon) in several hypervariable regions of the PRRSV genome in pigs as they progressed from acute to rebound infection. The selected regions are not only the most variable segments, but also play important roles in dictating host immune responses (Ansari et al., 2006; Beura et al., 2010; Sun et al., 2010, 2012; Vu et al., 2011). For example, nsp1α and nsp1β have strong inhibitory effects on beta interferon (IFN-β) promoter activation (Beura et al., 2010). Nsp2 is a key factor to antagonize type I IFN-related antiviral functions (Sun et al., 2010, 2012). Changes in N-glycosylation sites in GP3 and GP5 can alter the sensitivity of PRRSV to nAbs and help the virus escape host humoral immunity through glycan shielding (Ansari et al., 2006; Vu et al., 2011). In a previous study, we evaluated the makeup of PRRSV quasispecies in both SCID and normal pigs and found very few nucleotide substitutions in the structural genes, ORF2 through ORF7 during acute infection (Chen et al., 2015). In this study, we identified 14 nucleotide substitutions in the hypervariable regions of ORF3 and ORF5 during acute infection, whereas during rebound infection a total of 122 nucleotide

Fig. 5. Predicted peptide signal cleavage sites in GP5. Signal peptide cleavage prediction was performed using the web-based program, SignalP 4.0 (Petersen et al., 2011). The results show the C-scores for amino acid substitutions (underlined letters) shared by a majority of the four pigs. The predicted peptide cleavage site is shown by an arrow, which is located immediately in front of the amino acid with a high C-score.

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Table. 3 Amino acid substitutions under significantly positive selection in hypervariable regions of the PRRSV genome.

Sample

Inoculum 4 dpi sera 6576 6685 6721 6774 Mean 28 dpi sera 6576 6685 6721 6774 Mean 42 dpi sera 6576 6685 6721 6774 Mean P (Ω N 1)a P (Ω N 1)b

C338A (P–H) in nsp1

T839C (L–P) in nsp1

T2555C (L–S) in nsp2

A88G (T–A) in GP3

Depth

%

Depth

353

16

2855 1101 316 1265 1384 3248 684 621 713 1316

%

Depth

%

Depth

%

Depth

%

Depth

%

Depth

%

Depth

%

494

0

1129

0

498

0

497

44

528

39

320

0

320

0

17 34 11 14 19

877 1044 663 1284 967

0 0 0 0 0

1055 1175 295 1063 897

0 0 0 22 5

1420 684 1051 837 998

0 0 0 0 0

1301 627 814 774 879

37 57 31 48 43

1305 591 803 745 861

0 7 50 0 14

1121 548 1235 598 875

0 0 0 0 0

1118 545 1235 598 874

0 0 0 0 0

7 0 0 9 4

2571 733 733 834 1217

0 0 0 26 6

4073 718 653 299 1435

10 77 0 54 35

2365 473 850 222 977

0 0 46 14 15

2166 473 798 204 910

21 0 8 0 7

2079 416 773 197 866

0 0 0 0 0

1528 662 680 289 789

20 45 70 54 47

1338 633 661 259 722

0 23 6 14 11

7167 280 1072 631 2287 99⁎⁎ 99⁎⁎

0 0 30 0 7

380 944 359 280 490

97 0 98 0 49

569 644 465 437 528

0 31 0 99 32

519 595 436 420 492

0 26 0 0 6

492 561 407 401 465

0 0 0 0 0

450 118 426 243 309

70 0 42 70 45

452 118 421 242 308

0 0 52 62 29

955 0 482 0 1137 99 562 61 784 40 99⁎⁎ 100⁎⁎

99⁎⁎ 100⁎⁎

99⁎⁎ 99⁎⁎

C287T (P–L) in GP3

99⁎⁎ 100⁎⁎

T428C (F–S) in GP3

95⁎ 97⁎

C80T (A–V) in GP5

95⁎ 98⁎

A95G (N–S) in GP5

95⁎ 97⁎

a

BEB posterior probability from the model M2. BEB posterior probability from the model M8. ⁎ p ≥ 95%. ⁎⁎ p ≥ 99%. b

substitutions were found in the same regions (Figs. 2 and 3). During rebound infection, substitutions dramatically increased indicating that the adaptive immunity is likely an important part of the selective pressures driving high genetic diversity. Consistent with the notion that PRRSV has different replication sites in vivo, the quasispecies found in sera and tonsils were different. Thus,

Table 4 Nucleotide substitutions and percent frequency in tonsil versus serum in pigs 6576 and 6721 at 42 dpi.a Source Amplicon (location)

1-nsp1α (297–731) 2-nsp1β (713–1093)

3-nsp1β (997–1366) 4-nsp2 (1311–1725)

5-nsp2 (1705–2101)

6-nsp2 (2701–3040) 7-nsp2 (3535–3921) 8-ORF3 (12,761–13,154) 9-ORF5 (13,718–14,091)

Pig 6576

Pig 6721

Position

Serum

Tonsil

Serum

Tonsil

C227 C230 A587 A672 C871 A922 G1109 G1268 G1294 C1357 T1632 C1834 A1846 T25554 A2621 G2830 C3427 C255 C306 C372 G56 C80 A95 A243

T (55%) – – G (84%) – – – A (18%) A (17%) – – T (19%) G (17%) C (97%) G (12%) – – A (100%) T (17%) T (13%) A (29%) T (70%) – –

T (85%) – – G (74%) – – A (13%) A (15%) A (14%) A (25%) C (10%) T (12%) G (6%) C (99%) G (9%) A (24%) T (13%) A (100%) – T (19%) A (34%) T (35%) – –

– T (95%) G (37%) – T (33%) G (34%) – – – – – – – C (98%) – – – A (100%) – – – T (42%) G (52%) T (100%)

– T (24%) G (60%) – – – – – – – – – – C (97%) – – – A (100%) – – – T (62%) G (25%) T (92%)

a The nucleotide position numbers are shown relative to NVSL 97-7895. The locations of substitutions in nsp1α, nsp1β and nsp2 are relative to the ORF1a gene. The locations of substitutions in amplicons 8 and 9 are based on ORF3 and ORF5 start codons, respectively. Substitutions found in tonsil, but not serum or vice versa are shaded.

evaluating viral quasispecies only in sera may not detect mutations occurring during replication in organs such as the tonsil. Similar findings were reported for Hepatitis C virus (HCV) where distinct viral populations were identified in serum and liver samples (Blackard et al., 2014). It has been shown that various PRRSV isolates have distinct tissue tropism (Frydas et al., 2015). Our results suggest that lymphoid tissue tropism may play an important role in the selection of PRRSV populations capable of persisting in different lymphoid cells (Rowland et al., 2003; Rowland et al., 1999b). Independent evolution of viral quasispecies in lymphocytes of different tissues also helps to explain the high mutation rate and diversity of PRRSV. Our data suggested that, in order to investigate the genetic diversity of PRRSV populations in vivo, quasispecies in both sera and lymphoid organs should be assessed. Even though purifying (negative) selection continuously acted on hypervariable regions during acute and rebound infection, specific aa sites were determined to evolve under positive selection. These sites could serve as genetic markers for immune escaped viruses and some, if not all, may help virus variants to persist in pigs. For instance, we found two positively selected substitutions (A27V and N32S) in GP5 that were located at the edge of signal peptide cleavage sites. The A27V mutation resides at the beginning of the decoy epitope A (aa residues 27–31). The N32S substitution is located right after the decoy epitope and creates a new N-glycosylation site (Fig. 4). Mutations in the region could have several effects on the immunological properties of GP5. First, aa substitutions in the vicinity of the signal peptide sequence cleavage site could alter the length of the signal peptide. Second, the addition of serine residues could increase the number of N-linked glycosylation sites. Third, if epitope A is retained on the Nterminal end of GP5, antigenic variation can divert immunity from more conserved epitopes (Ostrowski et al., 2002; Thaa et al., 2013). Our results indicate that these positively selected sites likely contribute to immune evasion and PRRSV persistence by the following mechanisms: 1) Favoring the 26/27 cleavage site and retaining the decoy epitope in GP5 (Fig. 5), which is a presumed molecular mechanism of PRRSV persistence (Thaa et al., 2013). Additionall, the A27V mutation in the decoy epitope may help minimize and delay neutralizing antibody responses (Ostrowski et al., 2002). 2) The generation of a serine residue provides a site for potential glycosylation, which may

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affect disease persistence (Chackerian et al., 1994). 3) The new N-linked glycosylation site is located in the same region as previously identified N-glycosylation sites (Ansari et al., 2006; Vu et al., 2011). N-linked glycosylation sites in this region could help PRRSV evade the neutralizing antibody response by glycan shielding (Vu et al., 2011). Another report indicated that, within quasispecies virus variants with mutations in envelope glycoproteins that alter recognition by nAbs would be positively selected in situations when nAbs play an important role for virus control (Zinkernagel et al., 2001). The positively selected N-glycosylation site identified here may have a similar function. Besides the association between nAb response and virus rebound, several alternative hypothesis may be potential causes for viremia rebound. For instance, the rebound could arise from the heterogeneity of the virus distribution in various tissues within the host (Islam et al., 2013). Additionally, the positively selected mutants in quasispecies were not 100%, even not predominant (Table 3), suggesting that the selection occurred favoring the diversification of the viral population rather than selecting individual variants (Vignuzzi et al., 2006). To summarize, we developed an evolution model for quasispecies as PRRSV infection progresses from acute infection to virus rebound (Fig. S2). During acute infection (4 dpi), virus replicates efficiently in the absence of strong selective pressures from adaptive immunity, accumulating only a few variants. By 28 dpi, virus populations showed higher genetic diversity. Under increased pressure from adaptive immunity, the high PRRSV mutation rate and lack of proofreading and repair ability result in the accumulation of numerous variants. Adaptive immunity, including homologous nAb production, exerts increasingly greater negative selective pressure, resulting in decreased viremia and the extinction of the majority of virus mutants. However, by 42 dpi, a subset of viral variants has accumulated modifications that allow for survival in the presence of homologous nAb. Decreased selective pressures on the escaping mutants allow viral rebound until the immune system is able to mount another successful response, likely including heterologous neutralizing antibody. This proposed evolution model is consistent with a previous established mathematical model describing the relationship between the breadth of nAb response and viremia profile (Islam et al., 2013). In conclusion, for the first time we examined by deep sequencing the genetic variation of PRRSV quasispecies as the infection progressed from acute infection to virus rebound. PRRSV showed the highest genetic diversity at 28 dpi, additionally, different viral populations were identified in tonsils and sera collected at 42 dpi from the same pigs. In pigs showing virus rebound, PRRSV evolved under strong purifying selection. However, positively selected residues were also identified, which could serve as genetic markers and may contribute to immune evasion and persistence. Our findings suggest that adaptive immune pressure is important for generating high genetic diversity, and the generation of new mutants that gained fitness for survival under diversifying selection gives new insights into the understanding PRRSV persistence. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.03.002. Acknowledgments The authors would like to thank Dr. Xiangdong Li from the National Research Center for Veterinary Medicine (Luoyang, China) for his helpful advice during the revision of our manuscript. This work was funded by the National Pork Board Project #10-156, PRRS Host Genetics Consortium-Year 3. Dr. Chen is supported by the Scientific Research Foundation of Yangzhou University #137010925. References Allende, R., Laegreid, W.W., Kutish, G.F., Galeota, J.A., Wills, R.W., Osorio, F.A., 2000. Porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection. J. Virol. 74, 10834–10837.

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