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Porcine reproductive and respiratory syndrome virus replication and quasispecies evolution in pigs that lack adaptive immunity
Virus Research 195 (2015) 246–249
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Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Porcine reproductive and respiratory syndrome virus replication and quasispecies evolution in pigs that lack adaptive immunity Nanhua Chen a , Jack C.M. Dekkers b , Catherine L. Ewen a , Raymond R.R. Rowland a,∗ a b
Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States Department of Animal Science, Iowa State University, Ames, IA 50011-3150, United States
a r t i c l e
i n f o
Article history: Received 28 August 2014 Received in revised form 3 October 2014 Accepted 6 October 2014 Available online 14 October 2014 Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Severe combined immunodeficiency (SCID) Deep sequencing Quasispecies
a b s t r a c t The replication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in a line of pigs possessing a severe combined immunodeficiency (SCID). Real-time RT-PCR revealed a unique course of infection for the SCID group. During the course of infection, viremia was initially significantly lower than normal littermates, but by 21 days was significantly elevated. Deep sequencing of the viral structural genes at days 11 and 21 identified seven amino acid substitutions in both normal and SCID pigs. The most significant change was a W99 R substitution in GP2, which was present in the inoculum at a frequency of 35%, but eventually disappeared from all pigs regardless of immune status. Therefore, amino acid substitutions that appear during acute infection are likely the result of the adaptation of the virus to replication in pigs and not immune selection. © 2014 Elsevier B.V. All rights reserved.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly swine virus worldwide. The virus was first described in Europe in 1991 and subsequently isolated in the United States (Collins et al., 1992; Wensvoort et al., 1991). Based on comparisons of European and North American isolates, PRRSV is divided into Type 1 and Type 2 genotypes, respectively (Meng et al., 1995; Nelsen et al., 1999). Even though both genotypes appeared almost simultaneously and can produce similar clinical signs, they share only about 60–70% nucleotide identity (Hanada et al., 2005; Murtaugh et al., 2010). Nucleotide sequence differences within each genotype can vary as much as 20% at the level of ORF5 sequence (Lunney et al., 2010). The PRRSV genome contains a 5 untranslated region (UTR) followed by at least 10 open reading frames (ORFs), a 3 UTR, and a poly (A) tail. The 5 end of the genome is dominated by ORF1a and ORF1b proteins, pp1a and pp1ab, which are proteolytically cleaved to produce 14 non-structural proteins, responsible for the synthesis of genomic and subgenomic RNA, and modulation of innate immunity (Music and Gagnon, 2010). The 3 end of the genome, containing the PRRSV structural protein genes, ORFs 2-7, is translated from a nested set of six subgenomic mRNAs. The outer virion is composed of at least seven envelope proteins, GP2, E, GP3, GP4, GP5, ORF5a, and M. The major envelope proteins, GP5 and M, form
∗ Corresponding author. Tel.: +1 785 532 4631. E-mail address: [email protected] (R.R.R. Rowland). http://dx.doi.org/10.1016/j.virusres.2014.10.006 0168-1702/© 2014 Elsevier B.V. All rights reserved.
GP5-M heterodimer, while the minor glycoproteins, GP2, GP3 and GP4, form a heterotrimer that interacts with CD163 on the host cell (Das et al., 2010; Mardassi et al., 1996). The envelope surrounds the nucleocapsid, which is composed of nucleocapsid (N) protein dimers (Snijder et al., 2013). Recently, nsp2 was described as a virion-associated protein, but its functional role as a structural protein remains to be determined (Kappes et al., 2013). PRRSV is considered one of the most rapidly evolving viruses on the planet (Normile, 2007). Similar to other RNA viruses, PRRSV exists as a quasispecies of closely related sequences (Goldberg et al., 2003; Rowland et al., 1999). Antigenic and genetic drift are the likely mechanisms that explain the emergence of new genetic variants. Important consequences of genetic variation are the appearance of new viruses with enhanced virulence or with the capacity to escape vaccination (Chand et al., 2012; Tian et al., 2007). Even though immunological selection is considered a principal force driving the emergence of new viruses (Murtaugh et al., 2010), experimental evidence demonstrating the quantitative influence of adaptive immunity on the evolution of PRRSV is lacking. The recent characterization of a line of pigs with severe combined immunodeficiency (SCID) creates the opportunity to study the role of adaptive immunity in PRRSV replication and evolution. The SCID pig, first described by us, is markedly depleted in cells with markers associated with B and T cells (Ozuna et al., 2013) and fails to reject human tumors (Basel et al., 2012). In this study, we evaluated viremia and antibody responses in SCID and normal littermates during the first 21 days after PRRSV infection. Deep sequencing of
N. Chen et al. / Virus Research 195 (2015) 246–249
the PRRSV structural genes was used as a means to evaluate and compare the makeup of the viral quasispecies population in each group. Prior to initiating experiments involving animals and viruses, all work was approved by Kansas State University biological safety and animal care and use committees. Two litters were derived from the mating of SCID ± heterozygous parents. The presence of the SCID phenotype in the newborn piglets was based on relatively low numbers of total lymphocytes as determined by CBC measurements combined with the absence of immunoglobulin in in the serum. At the end of the study, the SCID phenotype was confirmed by histology (Ozuna et al., 2013). The two litters produced a total of 18 pigs, 12 normal and 6 with the SCID phenotype. Infection with PRRSV was performed at the Large Animal Research Center (Biosafety level 2), Kansas State University. No special housing conditions are required, since passive immunity from the dam provides protection for at least three to four weeks after weaning. At about three weeks of age, pigs were infected with a Type 2 PRRSV isolate, KS06-72109 (GenBank # KM252867). The virus inoculum was amplified by passage in MARC-145 cells, a simian cell line. Each pig was intramuscularly and intranasally inoculated with 105.0 TCID50 in two ml of MEM. Pigs were monitored daily for clinical signs and serum samples collected at 0, 4, 7, 11, 14 and 21 dpi. All samples were stored at −80 ◦ C until further use. Viremia in SCID and normal littermates was measured using the EZ-PRRSVTM MPX 4.0 Real Time RT-PCR Target-Specific Reagents (Tetracore® , Rockville, MD) and assays performed according to the manufacturer’s instructions. For standardization, each plate contained a set of Tetracore® Quantification Standards and Controls. All PCR reactions were carried out on a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in a 96-well format using the recommended cycling parameters. Results were reported as the log number of templates per 50 l PCR reaction. PRRSV viremia for the piglets is shown in Fig. 1A. The normal littermates showed a pattern of viremia typical of PRRSV infection: reaching a peak at about 11 dpi followed by a gradual decline. In contrast, the SCID group showed significantly lower amounts of virus in serum at 4, 7 and 11 dpi. At 11 dpi, the mean viremia for the SCID group was approximately one log lower than that of the normal group. However, instead of declining, viremia in the SCID group remained elevated, and by 21 dpi, was significantly higher than that of the normal littermates. Soon after, the experiment was terminated when the SCID pigs began to exhibit a variety of clinical signs, including weight loss and respiratory illness, a consequence of the lack of immunity. Anti-PRRSV immunity was assessed by fluorescent microsphere immunofluorescence (FMIA) or Luminex. Recombinant N protein was coupled to carboxylated Luminex MagPlex® polystyrene microspheres according to the manufacturer’s directions. Approximately 2500 antigen-coated beads, diluted in PBS with 10% goat serum (PBS-GS), were placed in each well of a 96-well polystyrene round bottom plate (Costar, Corning, NY). Fifty microliters of a 1:400 dilution of serum in PBS-GS was added to duplicate wells. The plate, wrapped in foil, was incubated for 30 min at room temperature with gentle shaking. Beads were washed three times with 190 l of PBS-GS. For the detection of IgG, biotin-SP-conjugated affinity purified goat anti-swine IgG was used (Jackson ImmunoResearch). IgM was detected with a biotin-labeled affinity-purified goat anti-swine IgM (KPL, Gaithersburg, MD). Secondary antibodies were diluted to 2 g/ml in PBS-GS and incubated with the microspheres for 30 min. Plates were washed three times, followed by the addition of 50 l of streptavidin-conjugated phycoerythrin (2 g/ml in PBS-GS; SAPE). After 30 min, the plates were washed and microspheres resuspended in 100 l of PBS-GS and analyzed on a MAGPIX instrument (Luminex) with Luminex® xPONENT 4.2 software. The results were reported as mean fluorescence intensity (MFI).
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Fig. 1. PRRSV viremia and antibody response for SCID pigs and normal littermates. (A) Viremia is shown as mean ± standard deviation. (B) Antibody results are shown as mean fluorescence intensity (MFI) ± standard deviation. P-values are shown for those days when there was a significant difference between groups. Statistics were performed using the Mann-Whitney U test. Results for both panels are for 12 normal pigs (solid line) and 6 SCID pigs (dashed line).
The antibody responses for normal and SCID pigs are shown in Fig. 1B. For normal pigs, IgM was first detected at 7 dpi, reached a peak at 11 dpi, and by 21 dpi approached background MFI values. This pattern is consistent with the primary antibody response of pigs to PRRSV infection (Molina et al., 2008). MFI values for all of the SCID pigs remained at background levels throughout the experiment. PRRSV-specific IgG was initially relatively high for all pigs, the result of a high non-specific level of background activity carried over by maternal antibody. For normal pigs, the initial decay in maternal antibody was followed by a steady increase in PRRSV-specific antibody. For the SCID group, the non-specific maternal antibody response continued to decay throughout the remainder of the experiment. By 21 dpi the MFI values for the SCID pigs approached background for the assay. These results confirmed that PRRSV-specific humoral immunity was absent in the PRRSVinfected SCID pigs.
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N. Chen et al. / Virus Research 195 (2015) 246–249
Table 1 Amino acid substitutions in PRRSV structural proteins at 11 and 21 days after infectiona Structural Protein Parent virus Day 11 Normal
Pig 1–10
Normal
2–1
SCID
1–4
SCID
1–9
SCID
2–2
SCID
2–5
SCID
2–8
Day 21 Normal
1–10
Normal
2–1
SCID
1–4
SCID
1–9
SCID
2–2
SCID
2–5
SCID
2–8
2 W99 R 35%(60)
2b R59 C 10%(63)
3 (91)
4 (108)
5 (129)
5a (129)
M (83)
N (75)
W99 R 3%(477) W99 R 8%(439) W99 R 9%(497) W99 R 10%(514) W99 R 15%(471) W99 R 27%(365) (366)
(502) (461) (517) (537) R59 C 10%(492) (381) (366)
(711) (376) (845) (94) (261) (391) (341)
(1528) (500) (264) V46 I 20%(415) I81 V 6%(302) (311) (281)
(866) (529) (1988) A121 V 28%(669) (449) (367) (296)
(866) (529) (868) (669) (449) (367) (296)
(638) (399) I95 V 66%(65) (350) (232) (337) (224)
(667) (586) (690) (541) (483) (454) (614)
(331) (325) (288) (295) (307) (298) (245)
(331) (325) (288) (295) (307) (298) (245)
(558) (274) (199) (260) (138) (255) (104)
(635) (220) (270) (9398) L171 F 12%(413) (350) (166)
(684) (389) (445) A121 V 90%(483) (455) (447) (229)
(684) (389) (445) (483) (455) (447) (229)
(326) (167) I95 V 86%(538) (269) I95 V 17%(315) (286) (106)
(300) (208) (359) (297) (286) (295) (324)
a The results show the amino acid substitutions in 2 normal pigs and 5 SCID pigs. The cutoff for detection of a mutation was based on a minimum variation frequency value of 5%. The percentage of the substitution in the population of sequences is below each substitution. Sequence coverage is shown in parentheses.
To analyze the makeup of viral quasispecies in SCID and normal littermates during acute infection, five pigs in the SCID group were selected for deep sequencing and, for the purpose of comparison, two normal littermates, one from each litter, were randomly selected. The two time points selected for investigation were 11 and 21 dpi, two time points when viremia was significantly elevated for normal and SCID groups, respectively. Eight primer sets directed at highly conserved regions were designed to amplify ORFs 2–7, the structural genes of the PRRSV genome. Total RNA was extracted from 100 l of tissue culture medium or serum using TRIzol® Reagent (Invitrogen). cDNA was prepared by reverse transcription using random hexamer primers and Transcriptor High Fidelity cDNA Synthesis Kit (Roche). As described by Daigle et al. (2011), two rounds of PCR amplification were used to prepare the library. Amplicon concentrations were normalized and mixed together in equal volumes to create a library containing 120 amplicons from all samples. The library was diluted to a final concentration of 1 × 107 molecules/l for each amplicon in 1 × TE buffer and stored at −20 ◦ C. The predicted sizes for the final products ranged between 340 bp and 446 bp. The amplicon library was sent to the Department of Plant Pathology, Kansas State University for emulsion PCR (emPCR) amplification and 454 sequencing. Lib-L emPCR Kit (Roche) was used for emPCR and the GS FLX Titanium Sequencing Kit XLR70 (Roche) was used for 454 sequencing according to the protocols described in the Amplification/Sequencing Method Manual. Sequencing was performed on a GS FLX+ System. Reads for each sample were sorted and mapped against the GenBank reference sequence (# KM252867) using 454 Life Sciences GS Reference Mapper (Version 2.6). Coverage (number of reads per amplicon) was calculated and variants were called. Variants were further filtered based on the coverage, variant frequency, and presence in a homopolymer region. Only high confidence single nucleotide
variants that had the following features were selected: (1) at least 3 non-duplicate reads with the nucleotide substitution; (2) a substitution frequency greater than 5%; and (3) not located within homopolymer regions. Mutations were further confirmed by sequence assembly visualization using Tablet (Milne et al., 2013). A summary of amino acid substitutions at 11 and 21 dpi for individual pigs is presented in Table 1. The coverage for each amplicon ranged between 60 and 1988 reads, with an average of 399 reads per amplicon. There were no insertions or deletions and only nine point mutations were identified, seven of which resulted in changes in amino acids. Overall, there was no difference in the pattern of amino acid substitutions between the SCID and normal littermate groups. A W99 R substitution was present in the virus inoculum at a frequency of 35%. By 11 dpi, the frequency of the W99 R substitution had substantially decreased, ranging from 0% (SCID pig 2–8) to 27% (SCID pig 2–5). By 21 dpi, the substitution was no longer detected. Examples of amino acids that increased in frequency can be found in the appearance of A121 V in GP5 in pig 1–9 and I95 V in M in pig 1–4, both SCID pigs. The A121 V substitution increased from 28% at 11 dpi to 90% at 21 dpi. The I95 V substitution increased from 66% (11 dpi) to 86% (21 dpi). At 21 dpi, the I95 V substitution appeared in a second SCID pig, 2–2. When adjusting the criteria below the 5% threshold, the I95 V substitution was detected in both normal and SCID pigs as well the parent virus (data not shown). Overall, these data show very few amino substitutions for the KS06 PRRSV isolate over the 21 days of infection. Furthermore, the same amino acid substitutions appeared in both normal and SCID pigs. The role of mutation and selection in preserving the pathogenesis or fitness of PRRSV has been studied by documenting nucleotide substitutions in viruses over time and by comparing the sequences of pathogenic and non-pathogenic variants of the same virus (Chang et al., 2002; Opriessnig et al., 2002; Yuan et al., 2001).
N. Chen et al. / Virus Research 195 (2015) 246–249
One outcome of the previous body of work is the notion that amino acid substitutions in structural proteins are the result of selective pressure by B and T cells (Murtaugh et al., 2010). One important consequence is antigenic drift, which contributes to long-term or “persistent” infection. In this study, we followed amino acid substitutions in ORFs 2–7 in pigs that lack B and T cells. The eventual disappearance of a W99 R substitution in GP2, present in the inoculum, occurred in both SCID pigs and normal littermates. This outcome demonstrated that the tryptophan substitution at position 99 was not the result of the escape from adaptive immunity. A more plausible explanation is the presence of Arg-99 in 35% of the viruses in the inoculum was the result of the selection of viruses better adapted for growth in MARC-145 cells, a simian cell line. The subsequent reversion to the wild-type sequence was due to the selection of viruses that “re-adapted” to replication in pigs. A molecular explanation can be found in the interaction of PRRSV with CD163, the receptor on the surface of macrophages (Calvert et al., 2007). GP2 and GP4 have been shown to directly interact with CD163, the PRRSV receptor (Das et al., 2010); however, the exact viral domains responsible for binding CD163 have not been determined. The selection of viruses with a tryptophan at position 99 of GP2 may increase the affinity of the virus for pig CD163. Similarly, the appearance of isoleucine-46, valine-81 and phenylalanine-171 in GP4 may also be driven by increased affinity for porcine CD163. None of the amino acid substitutions we identified occurred in known B-cell epitopes (aa 41-55 and 121-135 in GP2, and aa 51-65 in GP4) (de Lima et al., 2006). Compared to normal littermates, PRRSV replication followed a unique course in SCID pigs (Fig. 1A). The lower viremia in the SCID group at the early stage of infection suggests that fewer permissive macrophages are available for infection. Therefore, the role of T cells would be to regulate the number and/or permissiveness of macrophages for PRRSV infection. As demonstrated by Patton et al. (2009) and reviewed in (Cecere et al., 2012), T cell cytokines, such as IL-10, enhance the susceptibility of macrophages to infection. In contrast, T cell cytokines, such as IFN-␥, protect macrophages from infection (Rowland et al., 2001). Therefore, early in infection, T cells play a positive role in viremia by increasing the number of permissive macrophages. However, by Day 21 of infection, viremia in normal pigs has declined, while remaining high in SCIDs, indicating a failure of T cells to control virus replication. Acknowledgements Funding, wholly or in part, was provided by The National Pork Board (Grant #13-187). The SCID pigs were produced using funds from the Iowa State University Research Foundation and the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, U.S.A. (Project No. 3600). References Basel, M.T., Balivada, S., Beck, A.P., Kerrigan, M.A., Pyle, M.M., Dekkers, J.C., Wyatt, C.R., Rowland, R.R., Anderson, D.E., Bossmann, S.H., Troyer, D.L., 2012. Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs. Biores. Open Access 1 (2), 63–68. Calvert, J.G., Slade, D.E., Shields, S.L., Jolie, R., Mannan, R.M., Ankenbauer, R.G., Welch, S.K., 2007. CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses. J. Virol. 81 (14), 7371–7379. Cecere, T.E., Todd, S.M., Leroith, T., 2012. Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? Viruses 4 (5), 833–846. Chand, R.J., Trible, B.R., Rowland, R.R., 2012. Pathogenesis of porcine reproductive and respiratory syndrome virus. Curr. Opin. Virol. 2 (3), 256–263. Chang, C.C., Yoon, K.J., Zimmerman, J.J., Harmon, K.M., Dixon, P.M., Dvorak, C.M., Murtaugh, M.P., 2002. Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs. J. Virol. 76 (10), 4750–4763. Collins, J.E., Benfield, D.A., Christianson, W.T., Harris, L., Hennings, J.C., Shaw, D.P., Goyal, S.M., McCullough, S., Morrison, R.B., Joo, H.S., et al., 1992. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North
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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Porcine reproductive and respiratory syndrome virus replication and quasispecies evolution in pigs that lack adaptive immunity Nanhua Chen a , Jack C.M. Dekkers b , Catherine L. Ewen a , Raymond R.R. Rowland a,∗ a b
Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States Department of Animal Science, Iowa State University, Ames, IA 50011-3150, United States
a r t i c l e
i n f o
Article history: Received 28 August 2014 Received in revised form 3 October 2014 Accepted 6 October 2014 Available online 14 October 2014 Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Severe combined immunodeficiency (SCID) Deep sequencing Quasispecies
a b s t r a c t The replication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in a line of pigs possessing a severe combined immunodeficiency (SCID). Real-time RT-PCR revealed a unique course of infection for the SCID group. During the course of infection, viremia was initially significantly lower than normal littermates, but by 21 days was significantly elevated. Deep sequencing of the viral structural genes at days 11 and 21 identified seven amino acid substitutions in both normal and SCID pigs. The most significant change was a W99 R substitution in GP2, which was present in the inoculum at a frequency of 35%, but eventually disappeared from all pigs regardless of immune status. Therefore, amino acid substitutions that appear during acute infection are likely the result of the adaptation of the virus to replication in pigs and not immune selection. © 2014 Elsevier B.V. All rights reserved.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly swine virus worldwide. The virus was first described in Europe in 1991 and subsequently isolated in the United States (Collins et al., 1992; Wensvoort et al., 1991). Based on comparisons of European and North American isolates, PRRSV is divided into Type 1 and Type 2 genotypes, respectively (Meng et al., 1995; Nelsen et al., 1999). Even though both genotypes appeared almost simultaneously and can produce similar clinical signs, they share only about 60–70% nucleotide identity (Hanada et al., 2005; Murtaugh et al., 2010). Nucleotide sequence differences within each genotype can vary as much as 20% at the level of ORF5 sequence (Lunney et al., 2010). The PRRSV genome contains a 5 untranslated region (UTR) followed by at least 10 open reading frames (ORFs), a 3 UTR, and a poly (A) tail. The 5 end of the genome is dominated by ORF1a and ORF1b proteins, pp1a and pp1ab, which are proteolytically cleaved to produce 14 non-structural proteins, responsible for the synthesis of genomic and subgenomic RNA, and modulation of innate immunity (Music and Gagnon, 2010). The 3 end of the genome, containing the PRRSV structural protein genes, ORFs 2-7, is translated from a nested set of six subgenomic mRNAs. The outer virion is composed of at least seven envelope proteins, GP2, E, GP3, GP4, GP5, ORF5a, and M. The major envelope proteins, GP5 and M, form
∗ Corresponding author. Tel.: +1 785 532 4631. E-mail address: [email protected] (R.R.R. Rowland). http://dx.doi.org/10.1016/j.virusres.2014.10.006 0168-1702/© 2014 Elsevier B.V. All rights reserved.
GP5-M heterodimer, while the minor glycoproteins, GP2, GP3 and GP4, form a heterotrimer that interacts with CD163 on the host cell (Das et al., 2010; Mardassi et al., 1996). The envelope surrounds the nucleocapsid, which is composed of nucleocapsid (N) protein dimers (Snijder et al., 2013). Recently, nsp2 was described as a virion-associated protein, but its functional role as a structural protein remains to be determined (Kappes et al., 2013). PRRSV is considered one of the most rapidly evolving viruses on the planet (Normile, 2007). Similar to other RNA viruses, PRRSV exists as a quasispecies of closely related sequences (Goldberg et al., 2003; Rowland et al., 1999). Antigenic and genetic drift are the likely mechanisms that explain the emergence of new genetic variants. Important consequences of genetic variation are the appearance of new viruses with enhanced virulence or with the capacity to escape vaccination (Chand et al., 2012; Tian et al., 2007). Even though immunological selection is considered a principal force driving the emergence of new viruses (Murtaugh et al., 2010), experimental evidence demonstrating the quantitative influence of adaptive immunity on the evolution of PRRSV is lacking. The recent characterization of a line of pigs with severe combined immunodeficiency (SCID) creates the opportunity to study the role of adaptive immunity in PRRSV replication and evolution. The SCID pig, first described by us, is markedly depleted in cells with markers associated with B and T cells (Ozuna et al., 2013) and fails to reject human tumors (Basel et al., 2012). In this study, we evaluated viremia and antibody responses in SCID and normal littermates during the first 21 days after PRRSV infection. Deep sequencing of
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the PRRSV structural genes was used as a means to evaluate and compare the makeup of the viral quasispecies population in each group. Prior to initiating experiments involving animals and viruses, all work was approved by Kansas State University biological safety and animal care and use committees. Two litters were derived from the mating of SCID ± heterozygous parents. The presence of the SCID phenotype in the newborn piglets was based on relatively low numbers of total lymphocytes as determined by CBC measurements combined with the absence of immunoglobulin in in the serum. At the end of the study, the SCID phenotype was confirmed by histology (Ozuna et al., 2013). The two litters produced a total of 18 pigs, 12 normal and 6 with the SCID phenotype. Infection with PRRSV was performed at the Large Animal Research Center (Biosafety level 2), Kansas State University. No special housing conditions are required, since passive immunity from the dam provides protection for at least three to four weeks after weaning. At about three weeks of age, pigs were infected with a Type 2 PRRSV isolate, KS06-72109 (GenBank # KM252867). The virus inoculum was amplified by passage in MARC-145 cells, a simian cell line. Each pig was intramuscularly and intranasally inoculated with 105.0 TCID50 in two ml of MEM. Pigs were monitored daily for clinical signs and serum samples collected at 0, 4, 7, 11, 14 and 21 dpi. All samples were stored at −80 ◦ C until further use. Viremia in SCID and normal littermates was measured using the EZ-PRRSVTM MPX 4.0 Real Time RT-PCR Target-Specific Reagents (Tetracore® , Rockville, MD) and assays performed according to the manufacturer’s instructions. For standardization, each plate contained a set of Tetracore® Quantification Standards and Controls. All PCR reactions were carried out on a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in a 96-well format using the recommended cycling parameters. Results were reported as the log number of templates per 50 l PCR reaction. PRRSV viremia for the piglets is shown in Fig. 1A. The normal littermates showed a pattern of viremia typical of PRRSV infection: reaching a peak at about 11 dpi followed by a gradual decline. In contrast, the SCID group showed significantly lower amounts of virus in serum at 4, 7 and 11 dpi. At 11 dpi, the mean viremia for the SCID group was approximately one log lower than that of the normal group. However, instead of declining, viremia in the SCID group remained elevated, and by 21 dpi, was significantly higher than that of the normal littermates. Soon after, the experiment was terminated when the SCID pigs began to exhibit a variety of clinical signs, including weight loss and respiratory illness, a consequence of the lack of immunity. Anti-PRRSV immunity was assessed by fluorescent microsphere immunofluorescence (FMIA) or Luminex. Recombinant N protein was coupled to carboxylated Luminex MagPlex® polystyrene microspheres according to the manufacturer’s directions. Approximately 2500 antigen-coated beads, diluted in PBS with 10% goat serum (PBS-GS), were placed in each well of a 96-well polystyrene round bottom plate (Costar, Corning, NY). Fifty microliters of a 1:400 dilution of serum in PBS-GS was added to duplicate wells. The plate, wrapped in foil, was incubated for 30 min at room temperature with gentle shaking. Beads were washed three times with 190 l of PBS-GS. For the detection of IgG, biotin-SP-conjugated affinity purified goat anti-swine IgG was used (Jackson ImmunoResearch). IgM was detected with a biotin-labeled affinity-purified goat anti-swine IgM (KPL, Gaithersburg, MD). Secondary antibodies were diluted to 2 g/ml in PBS-GS and incubated with the microspheres for 30 min. Plates were washed three times, followed by the addition of 50 l of streptavidin-conjugated phycoerythrin (2 g/ml in PBS-GS; SAPE). After 30 min, the plates were washed and microspheres resuspended in 100 l of PBS-GS and analyzed on a MAGPIX instrument (Luminex) with Luminex® xPONENT 4.2 software. The results were reported as mean fluorescence intensity (MFI).
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Fig. 1. PRRSV viremia and antibody response for SCID pigs and normal littermates. (A) Viremia is shown as mean ± standard deviation. (B) Antibody results are shown as mean fluorescence intensity (MFI) ± standard deviation. P-values are shown for those days when there was a significant difference between groups. Statistics were performed using the Mann-Whitney U test. Results for both panels are for 12 normal pigs (solid line) and 6 SCID pigs (dashed line).
The antibody responses for normal and SCID pigs are shown in Fig. 1B. For normal pigs, IgM was first detected at 7 dpi, reached a peak at 11 dpi, and by 21 dpi approached background MFI values. This pattern is consistent with the primary antibody response of pigs to PRRSV infection (Molina et al., 2008). MFI values for all of the SCID pigs remained at background levels throughout the experiment. PRRSV-specific IgG was initially relatively high for all pigs, the result of a high non-specific level of background activity carried over by maternal antibody. For normal pigs, the initial decay in maternal antibody was followed by a steady increase in PRRSV-specific antibody. For the SCID group, the non-specific maternal antibody response continued to decay throughout the remainder of the experiment. By 21 dpi the MFI values for the SCID pigs approached background for the assay. These results confirmed that PRRSV-specific humoral immunity was absent in the PRRSVinfected SCID pigs.
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Table 1 Amino acid substitutions in PRRSV structural proteins at 11 and 21 days after infectiona Structural Protein Parent virus Day 11 Normal
Pig 1–10
Normal
2–1
SCID
1–4
SCID
1–9
SCID
2–2
SCID
2–5
SCID
2–8
Day 21 Normal
1–10
Normal
2–1
SCID
1–4
SCID
1–9
SCID
2–2
SCID
2–5
SCID
2–8
2 W99 R 35%(60)
2b R59 C 10%(63)
3 (91)
4 (108)
5 (129)
5a (129)
M (83)
N (75)
W99 R 3%(477) W99 R 8%(439) W99 R 9%(497) W99 R 10%(514) W99 R 15%(471) W99 R 27%(365) (366)
(502) (461) (517) (537) R59 C 10%(492) (381) (366)
(711) (376) (845) (94) (261) (391) (341)
(1528) (500) (264) V46 I 20%(415) I81 V 6%(302) (311) (281)
(866) (529) (1988) A121 V 28%(669) (449) (367) (296)
(866) (529) (868) (669) (449) (367) (296)
(638) (399) I95 V 66%(65) (350) (232) (337) (224)
(667) (586) (690) (541) (483) (454) (614)
(331) (325) (288) (295) (307) (298) (245)
(331) (325) (288) (295) (307) (298) (245)
(558) (274) (199) (260) (138) (255) (104)
(635) (220) (270) (9398) L171 F 12%(413) (350) (166)
(684) (389) (445) A121 V 90%(483) (455) (447) (229)
(684) (389) (445) (483) (455) (447) (229)
(326) (167) I95 V 86%(538) (269) I95 V 17%(315) (286) (106)
(300) (208) (359) (297) (286) (295) (324)
a The results show the amino acid substitutions in 2 normal pigs and 5 SCID pigs. The cutoff for detection of a mutation was based on a minimum variation frequency value of 5%. The percentage of the substitution in the population of sequences is below each substitution. Sequence coverage is shown in parentheses.
To analyze the makeup of viral quasispecies in SCID and normal littermates during acute infection, five pigs in the SCID group were selected for deep sequencing and, for the purpose of comparison, two normal littermates, one from each litter, were randomly selected. The two time points selected for investigation were 11 and 21 dpi, two time points when viremia was significantly elevated for normal and SCID groups, respectively. Eight primer sets directed at highly conserved regions were designed to amplify ORFs 2–7, the structural genes of the PRRSV genome. Total RNA was extracted from 100 l of tissue culture medium or serum using TRIzol® Reagent (Invitrogen). cDNA was prepared by reverse transcription using random hexamer primers and Transcriptor High Fidelity cDNA Synthesis Kit (Roche). As described by Daigle et al. (2011), two rounds of PCR amplification were used to prepare the library. Amplicon concentrations were normalized and mixed together in equal volumes to create a library containing 120 amplicons from all samples. The library was diluted to a final concentration of 1 × 107 molecules/l for each amplicon in 1 × TE buffer and stored at −20 ◦ C. The predicted sizes for the final products ranged between 340 bp and 446 bp. The amplicon library was sent to the Department of Plant Pathology, Kansas State University for emulsion PCR (emPCR) amplification and 454 sequencing. Lib-L emPCR Kit (Roche) was used for emPCR and the GS FLX Titanium Sequencing Kit XLR70 (Roche) was used for 454 sequencing according to the protocols described in the Amplification/Sequencing Method Manual. Sequencing was performed on a GS FLX+ System. Reads for each sample were sorted and mapped against the GenBank reference sequence (# KM252867) using 454 Life Sciences GS Reference Mapper (Version 2.6). Coverage (number of reads per amplicon) was calculated and variants were called. Variants were further filtered based on the coverage, variant frequency, and presence in a homopolymer region. Only high confidence single nucleotide
variants that had the following features were selected: (1) at least 3 non-duplicate reads with the nucleotide substitution; (2) a substitution frequency greater than 5%; and (3) not located within homopolymer regions. Mutations were further confirmed by sequence assembly visualization using Tablet (Milne et al., 2013). A summary of amino acid substitutions at 11 and 21 dpi for individual pigs is presented in Table 1. The coverage for each amplicon ranged between 60 and 1988 reads, with an average of 399 reads per amplicon. There were no insertions or deletions and only nine point mutations were identified, seven of which resulted in changes in amino acids. Overall, there was no difference in the pattern of amino acid substitutions between the SCID and normal littermate groups. A W99 R substitution was present in the virus inoculum at a frequency of 35%. By 11 dpi, the frequency of the W99 R substitution had substantially decreased, ranging from 0% (SCID pig 2–8) to 27% (SCID pig 2–5). By 21 dpi, the substitution was no longer detected. Examples of amino acids that increased in frequency can be found in the appearance of A121 V in GP5 in pig 1–9 and I95 V in M in pig 1–4, both SCID pigs. The A121 V substitution increased from 28% at 11 dpi to 90% at 21 dpi. The I95 V substitution increased from 66% (11 dpi) to 86% (21 dpi). At 21 dpi, the I95 V substitution appeared in a second SCID pig, 2–2. When adjusting the criteria below the 5% threshold, the I95 V substitution was detected in both normal and SCID pigs as well the parent virus (data not shown). Overall, these data show very few amino substitutions for the KS06 PRRSV isolate over the 21 days of infection. Furthermore, the same amino acid substitutions appeared in both normal and SCID pigs. The role of mutation and selection in preserving the pathogenesis or fitness of PRRSV has been studied by documenting nucleotide substitutions in viruses over time and by comparing the sequences of pathogenic and non-pathogenic variants of the same virus (Chang et al., 2002; Opriessnig et al., 2002; Yuan et al., 2001).
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One outcome of the previous body of work is the notion that amino acid substitutions in structural proteins are the result of selective pressure by B and T cells (Murtaugh et al., 2010). One important consequence is antigenic drift, which contributes to long-term or “persistent” infection. In this study, we followed amino acid substitutions in ORFs 2–7 in pigs that lack B and T cells. The eventual disappearance of a W99 R substitution in GP2, present in the inoculum, occurred in both SCID pigs and normal littermates. This outcome demonstrated that the tryptophan substitution at position 99 was not the result of the escape from adaptive immunity. A more plausible explanation is the presence of Arg-99 in 35% of the viruses in the inoculum was the result of the selection of viruses better adapted for growth in MARC-145 cells, a simian cell line. The subsequent reversion to the wild-type sequence was due to the selection of viruses that “re-adapted” to replication in pigs. A molecular explanation can be found in the interaction of PRRSV with CD163, the receptor on the surface of macrophages (Calvert et al., 2007). GP2 and GP4 have been shown to directly interact with CD163, the PRRSV receptor (Das et al., 2010); however, the exact viral domains responsible for binding CD163 have not been determined. The selection of viruses with a tryptophan at position 99 of GP2 may increase the affinity of the virus for pig CD163. Similarly, the appearance of isoleucine-46, valine-81 and phenylalanine-171 in GP4 may also be driven by increased affinity for porcine CD163. None of the amino acid substitutions we identified occurred in known B-cell epitopes (aa 41-55 and 121-135 in GP2, and aa 51-65 in GP4) (de Lima et al., 2006). Compared to normal littermates, PRRSV replication followed a unique course in SCID pigs (Fig. 1A). The lower viremia in the SCID group at the early stage of infection suggests that fewer permissive macrophages are available for infection. Therefore, the role of T cells would be to regulate the number and/or permissiveness of macrophages for PRRSV infection. As demonstrated by Patton et al. (2009) and reviewed in (Cecere et al., 2012), T cell cytokines, such as IL-10, enhance the susceptibility of macrophages to infection. In contrast, T cell cytokines, such as IFN-␥, protect macrophages from infection (Rowland et al., 2001). Therefore, early in infection, T cells play a positive role in viremia by increasing the number of permissive macrophages. However, by Day 21 of infection, viremia in normal pigs has declined, while remaining high in SCIDs, indicating a failure of T cells to control virus replication. Acknowledgements Funding, wholly or in part, was provided by The National Pork Board (Grant #13-187). The SCID pigs were produced using funds from the Iowa State University Research Foundation and the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, U.S.A. (Project No. 3600). References Basel, M.T., Balivada, S., Beck, A.P., Kerrigan, M.A., Pyle, M.M., Dekkers, J.C., Wyatt, C.R., Rowland, R.R., Anderson, D.E., Bossmann, S.H., Troyer, D.L., 2012. Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs. Biores. Open Access 1 (2), 63–68. Calvert, J.G., Slade, D.E., Shields, S.L., Jolie, R., Mannan, R.M., Ankenbauer, R.G., Welch, S.K., 2007. CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses. J. Virol. 81 (14), 7371–7379. Cecere, T.E., Todd, S.M., Leroith, T., 2012. Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? Viruses 4 (5), 833–846. Chand, R.J., Trible, B.R., Rowland, R.R., 2012. Pathogenesis of porcine reproductive and respiratory syndrome virus. Curr. Opin. Virol. 2 (3), 256–263. Chang, C.C., Yoon, K.J., Zimmerman, J.J., Harmon, K.M., Dixon, P.M., Dvorak, C.M., Murtaugh, M.P., 2002. Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs. J. Virol. 76 (10), 4750–4763. Collins, J.E., Benfield, D.A., Christianson, W.T., Harris, L., Hennings, J.C., Shaw, D.P., Goyal, S.M., McCullough, S., Morrison, R.B., Joo, H.S., et al., 1992. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North
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