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The Journal of Immunology, 2007, 178, 6320 -6331
Copyright © 2007 by The American Association of Immunologists, Inc.
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Antibody Repertoire Development in Fetal and Neonatal Piglets: XIX. Undiversified B Cells with Hydrophobic HCDR3s Preferentially Proliferate in the Porcine Reproductive and Respiratory Syndrome1

John E. Butler2,*, Caitlin D. Lemke{dagger}, Patrick Weber*, Marek Sinkora{ddagger} and Kelly M. Lager§

* Department of Microbiology and Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242; {dagger} Robarts Research Institute, London, Ontario, Canada; {ddagger} Czech Academy of Science, Novi Hradek, Czech Republic; and § Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, U.S. Department of Agriculture-Agricultural Research Service, Ames, IA 50010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Porcine respiratory and reproductive syndrome virus (PRRSV) causes an extraordinary increase in the proportion of B cells resulting in lymphoid hyperplasia, hypergammaglobulinemia, and autoimmunity in neonatal piglets. Spectratypic analysis of B cells from neonatal isolator piglets show a non-Gaussian pattern with preferential expansion of clones bearing certain H chain third complementary region (HCDR3) lengths. However, only in PRRSV-infected isolator piglets was nearly the identical spectratype observed for all lymphoid tissues. This result suggests dissemination of the same dominant B cell clones throughout the body. B cell expansion in PRRS was not associated with preferential VH gene usage or repertoire diversification and these cells appeared to bear a naive phenotype. The B cell population observed during infection comprised those with hydrophobic HCDR3s, especially sequences encoded by reading frame 3 of DHA that generates the AMVLV motif. Thus, the hydropathicity profile of B cells after infection was skewed to favor those with hydrophobic binding sites, whereas the normally dominant region of the hydropathicity profile containing neutral HCDR3s was absent. We believe that the hypergammaglobulinemia results from the products of these cells. We speculate that PRRSV infection generates a product that engages the BCR of naive B cells, displaying the AMVLV and similar motifs in HCDR3 and resulting in their T-independent proliferation without repertoire diversification.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Viral infection impacts the immune system in many different ways. Some infections evoke a robust Ab response that neutralizes further infectivity. Others promote a more delayed cytotoxic response that eliminates infected cells whereas some do both. Many viral pathogens have evolved mechanisms to down-regulate MHC class I presentation of viral Ags. Some produce mimic FcRs and others alter cytokine profiles (1, 2, 3). A few viruses elaborate T cell superantigens (SAgs)3 that expand T cell subpopulations independent of virus specificity that can result in cytokine shock and cause clonal deletion (4, 5). Some viruses elaborate B cell SAgs that expand nonvirus-specific B cell subsets (6). This effect may explain why lymphocytic choriomeningitis virus and other viruses evoke polyclonal B cell activation that can result in autoantibodies (7, 8, 9, 10, 11).

We reported that infection of Caesarian-derived isolator piglets with porcine respiratory and reproductive syndrome (PRRS) virus (PRRSV) resulted in polyclonal B cell activation, autoimmunity, extreme lymphoid hyperplasia, and hypergammaglobulinemia but few virus-specific Abs (12). A similar polyclonal syndrome is produced in mice by lactate dehydrogenase-elevating virus, a related virus to PRRSV (9, 11, 12, 13). We used PRRSV infection in isolator piglets as a model to understand polyclonal B cell activation and autoimmunity associated with viral infections.

PRRSV is a positive-strand RNA virus of the arterivirus group (14, 15, 16, 17). The disease syndrome (also called swine infertility and respiratory syndrome, mystery swine disease, and "blue ear" disease) initially manifested in sows and was characterized by increased rates of reproductive failure, including anorexia, abortions, early farrowings, stillbirths, mummified fetuses, weak newborn piglets, and a delayed return to estrus (reviewed in Ref. 18). PRRSV Ags are localized primarily in porcine alveolar macrophages in infected piglets (19, 20), suggesting that PRRSV replicates in vivo in porcine alveolar macrophages and in other macrophages and dendritic cells throughout the body (21, 22). On average, 40–80% of herds are infected by PRRSV in the United States and Europe (18, 23). PRRS is currently considered the most important infectious disease of swine worldwide, resulting in an estimated $560 million annual loss in the United States (24).

The adaptive immune response to PRRSV has not been well defined and is both controversial and contradictory. PRRS is associated with a relative decrease in CD4+ cells and an increase in CD8+ cells in conventional and specific pathogen-free piglets (25). CD4+ cell numbers in blood remained unchanged for 35 days postinfection (dpi), whereas after 21 dpi both CD8+ and IgM+ cell numbers proportionally increase. An increase in the numbers of B cells and CD8+ cells in bronchoalveolar lavage (BAL) can be seen as early as 5 dpi without effect on CD4+ cells (26, 27). PRRSV-specific T cell responses appear delayed and are not detected until 28 days dpi (28, 29). Blocking studies indicate the delayed T cell response is largely due to a delayed response of CD4+ T cells (28, 29). Depletion of CD8+ cells had little effect on clinical symptoms and, not surprisingly, on PRRSV-specific Abs (30). By comparison swine influenza (SIV)-specific T cells are detected within 7 dpi (31). Although SIV induces a significant inflammatory response in the respiratory tract that appears related to viral clearance (32), no such inflammation is associated with PRRS (32, 33). Despite a delay in virus-specific T cell responses, Ab responses to PRRSV of all major isotypes occur 6–14 dpi in blood or BAL (34, 35, 36). IL-10 is up-regulated (37, 38, 39), but the appearance of virus-neutralizing (VN) Abs and CD4+ antiviral responses is delayed until 28 dpi (40). When present, VN is often mediated by Abs to open reading frame (ORF)5 glycoprotein (41, 42) and to a lesser extent epitopes on the M protein (membrane protein, ORF6;43), even through robust responses to this highly immunogenic nucleocapsid protein (N protein, ORF7) are seen (44, 45). Because PRRSV causes polyclonal B cell activation, the early non-VN Abs in PRRS may represent a T-independent preadaptive response triggered through pathogen-associated molecular patterns. These may nevertheless play a role in reducing the viral load (34). Of major concern to animal health is that PRRSV 1) results in persistent infections that may require 150 days to clear (46) and 2) infections increase susceptibility to secondary infection and may interfere with the success of vaccines (47, 48, 49).

Our recent observation of hypergammaglobulinemia (100- to 1,000-fold increase in serum IgG; 10- to 100-fold increase in IgA and IgM in PRRSV-infected isolator piglets (12)) is consistent with immune dysregulation. This hypergammaglobulinemia and the delay in Th2 responses may play a role in virus persistence. Although B cell expansion during PRRSV infection is polyclonal, spectratypic analyses consistently showed that BCRs with certain H chain CDR3 lengths (H chain third complementary region; HCDR3) often dominate the profile (12). Because of the limited combinatorial diversity in swine (50), HCDR3 length is a quasi-clone marker. Therefore, it suggests that PRRSV causes selective expansion of certain B cell clones, although the low antiviral titer suggests that few of these clones are virus specific. We show here that the expanded B cell population in PRRS is not associated with preferential usage of a VH gene or typical repertoire diversification, but that >80% selectively used reading frame (RF)3 of diversity segment A (RF3-DHA) in germline configuration yielding an unusually hydrophobic binding site motif, AMVLV. Meanwhile, the normal neutral HCDR3 hydrophobicity profile fails to develop. We propose that PRRSV targets for proliferation B cells of the preimmune repertoire that bear this and very similar HCDR3 motifs. We further suggest that PRRSV infections are persistent because they dysregulate the immune system through T-independent expansion of a naive B cell subpopulation that is not virus-specific, which in turn delays stimulation of the virus-specific CD4 T cells needed for effective VN Abs. It is also possible that Igs with hydrophobic HCDR3s may form aggregates that accumulate in the glomeruli of PRRSV-infected piglets and may even contribute to the autoantibodies we have described (12), collectively causing damage that weakens resistance to opportunistic pathogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal studies

All animal studies were conducted at the National Animal Disease Center (NADC; Ames, IA). Piglets were recovered by closed hysterectomy from 112-day gravid outbred swine, placed in groups of four in rigid tub isolators, and reared on ESPLac (PetAg) as described previously (12, 51). All studies were approved by the Animal Care and Use Committee of the NADC (Ames, IA). Most animals remained germfree whereas others were colonized with benign Escherichia coli as described previously (52), although we previously showed that E. coli colonization did not affect our results (12). Groups of three to five piglets from the same litter were used and were treated one of three ways: 1) inoculated intranasally with 104 TCID50 PRRSV; 2) inoculated intranasally with 104 TCID50 SIV; or 3) given a sham inoculum. All inoculations took place on day 7 (0 dpi). Animals were maintained for up to 46 days postinoculation (dpi) but most were necropsied at 10 and 25 dpi. A number of piglets were euthanized at delivery to obtain tissues for determining baseline data. Mesenteric lymph nodes (MLN) and PBLs from conventional young pigs were provided by Dr. J. Urban, Jr. (U.S. Department of Agriculture-Agricultural Research Service, Beltsville, MD).

Sample collection

Blood samples were collected weekly from the time of birth (–7 dpi) for preparation of serum and for recovery of PBLs. The former were used for measurement of Ig levels as described previously (12, 51). The latter were used for CBC analyses and for flow cytometric analysis (FCM), and the remainder were frozen in liquid nitrogen for eventual transcript analysis. Animals were euthanized with pentobarbital (Sleepaway; Fort Dodge Laboratories). At necropsy, lungs were lavaged and used to recover cells for FCM and transcript analysis. Various lymphoid tissues were also collected, frozen in liquid nitrogen, and stored at –70°C for transcript analysis. These tissues included the following: tonsil (TON), tracheal-bronchial lymph nodes (BLN), lung, inguinal lymph nodes, MLN, spleen (Spl), ileal Peyer’s patches (IPP), and jejunal Peyer’s patches.

BAL was centrifuged at 1000 x g for 15 min and the supernatant recovered for later Ig and protein analysis. The BAL cell pellet was suspended in TRIzol and frozen at –20°C. Blood collected in anticoagulation tubes was processed as previously described for the recovery of PBMC and plasma (12, 52, 53). The plasma was used for Ig determinations and was cultured for virus as described previously (54).

Flow cytometric analysis

FCM to obtain preliminary phenotypic data was done using a FACSCalibur flow cytometer (BD Biosciences). Three-color FCM was performed as described previously (53).

Spectratypic analysis

The HCDR3 plays a significant role in Ag recognition and binding (55, 56). HCDR3 is especially important in swine because of their limited combinatorial diversity (50, 57, 58). Because single B cell clones display a single-length HCDR3 (Ref. 59 and Fig. 1A; L23 and L35 cells), separation of HCDR3 regions on polyacrylamide sequencing gels provides a clonotypic analysis known as spectratyping. Rearranged VDJs and HCDR3 were recovered from solid tissue preserved at –70°C and those stored in TRIzol. These were processed as previously described for the preparation of total RNA and subsequently cDNA (12, 60). VDJs expressed on IgM and IgG transcripts were recovered as described previously (60, 61). HCDR3 regions for spectratypic analysis of Ig were amplified using a FR3-JH primer set as described previously (50). However, in this study, acrylamide gels were not totally dried but coated in saran wrap and incubated overnight at 4°C with Kodak BioMax MR film. The autoradiograph was subsequently aligned with the partially dried original gel, and prominent HCDR3 polynucleotides (pnt) (Figs. 1–3) were then excised from the gel, cloned, and sequenced.


Figure 1
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  		FIGURE 1. Spectratypic analyses of B cells from various tissues at 10 dpi in PRRSV-infected piglets. HCDR3 nucleotide length standards are indicated. A, PRRS piglet 49B; B, PRRS piglet 49C. The spectratypes of the MLN of three conventionally reared young pigs (PIC) are shown on the far right. The spectratype of HCDR3 from porcine B cell lines L23 and L35 with single pnt bands at 15 are also shown. B, Blank lane. Arrows denote prominently expressed HCDR3 lengths. Col, Colon; ILN, inguinal lymph node; jPP, jejunal Peyer’s patches; PBL, PBLs indicated as collected on 0, 7, and 10 dpi; Lg, lung parenchyma; BLN.

 

Figure 2
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  		FIGURE 2. Comparative spectratype analyses of B cells from various tissues of PRRSV-infected piglets at 25 dpi. A, Spectratypic analyses of HCDR3 from piglet 49D. The abbreviations for the different tissues are the same as in Fig. 1. Arrows call attention to prominent pnts. Unlabeled lanes are blanks. B, Comparative spectratypic profiles of three PRRSV-infected animals in four different lymphoid tissues. Arrows indicate prominent HCDR3 pnt lengths that are responsible for the tissue-wide spectratype of each animal.

 

Figure 3
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  		FIGURE 3. Spectratypic analysis of HCDR3 from SIV-infected and sham control piglets at 25 dpi. A, HCDR3 spectratype of SIV-infected piglet 48A. B, HCDR3 spectratype of a sham control piglet. Legend is similar to Fig. 1. The brackets emphasize spectratypes that are similar among tissues. The spectratype of newborns (data not shown) resembles the Gaussian spectratype of IPP and thymus shown in this figure (50 ). Lg, lung; jPP, jejunal Peyer’s patches.

 
Sequence analysis

HCDR3 pnts of predominant HCDR3 lengths (e.g., 27, 39, 42, 45, 59 and Fig. 1) were recovered by removal of gel segments using a scalpel and placed into 100 µl of dH2O. Because pnt bands having a total length of 36–45 were often dominant in the spectratype of PRRSV-infected piglets and in controls (Figs. 1–3), these were targeted for recovery and sequence analysis as well as a few other prominent lengths that were occasionally observed. These were incubated overnight to permit diffusion of the DNA, and the fluid phase was targeted for 15 cycles of PCR using the same primer set used for original amplification of HCDR3. PCR products of the expected length were verified by agarose electrophoresis, and the resulting products were cloned into pCR2 TOPO (Invitrogen Life Technologies). Plasmid DNA was sequenced in the University of Iowa DNA core using the four-color ABI PRISM DNA Analyzer (Applied Biosystems). Amino acid sequences were deduced and analyzed in the Omiga program (Accelrys) for the possession of unique and shared motifs and for identification of DH usage. The frequency of recovery of identical sequences was recorded for each treatment group (see Table I). The deduced amino acid sequence of the various CDR3 regions extending from, but not including, the FR3 cysteine at position 104 down to the 5' region of JH, but not including the invariant tryptophan that starts FR4, were analyzed as described by Kyte and Doolittle (62) using the principles described by Eisenberg (63) as applied to HCDR3 sequences by Ivanov et al. (64). HCDR3 sequences recovered from C{gamma} transcripts from PRRSV-infected piglets were compared with those from sham controls, SIV-infected piglets, colonized isolator piglets, and conventionally reared parasite-infected young pigs (PIC) (50, 61). Sequence data are based on >415 transcript, 60–100 for each treatment group. We focused on IgG transcripts so that the number of sequences per group would be large enough to avoid bias from limited sample numbers. Furthermore, because the hypergammaglobulinemia and Ig deposition we observed in PRRSV-infected animals involves IgG, IgM, and IgA (12), and the ratio of their concentration (40:3:1) is similar to that in the blood of newborns (30:3:1) (60), we reasoned that IgG transcripts were representative of all major isotypes in this disease.


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  		Table I. Molecular characteristics of major HCDR3 lengths recovered from different treatment groups

 
VH gene usage

VH gene usage by IgM and IgG transcripts from PRRSV-infected piglets and sham controls was determined as described elsewhere (58, 61, 65). Briefly, rearranged VDJs transcribed with IgM and IgG were recovered from cDNA by PCR. The size of the product was verified by agarose electrophoresis, and the appropriate pnt band was removed for secondary amplification. Following secondary amplification the resulting product was cloned into pCR2 TOPO (Invitrogen Life Technologies). VH gene usage was determined by clonal hybridization using probes specific for the CDR1 and CDR2 region of the major porcine VH gene (58, 61, 65).

Statistical analysis

Statistical analysis of data when appropriate was done using the stats program of Prism (GraphPad), with the help of Dr. K. Chaloner (Department of Biostatistics, University of Iowa, Iowa City, IA). ANOVA was used to compare the frequency of recovery of identical HCDR3 sequences. Simple mean differences were compared by two-sided Student’s t analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PRRSV infection results in a spectratype shared among tissues

Fig. 1A compares the HCDR3 spectratype of B cells in various tissues of PRRSV-infected piglet 49B at 10 dpi. Although the profile is polyclonal like that in PIC MLN (Fig. 1, A and B, far right), certain pnt bands are pronounced, e.g., pnt 27,42 and 57, in nearly all tissues examined. A similar HCDR3 spectratypic profile, in which pnt 42 is pronounced, is also shared among tissues from PRRSV-infected piglet 49C (Fig. 1B). The spectratype of the PBLs at both 7 and 10 dpi in both animals is somewhat less similar to the pattern in other tissues, but the pronounced bands are still apparent. The pattern at 0 dpi (Fig. 1B) resembles the polyclonal pattern of the control MLN. Fig. 2A shows the spectratype of a third PRRSV-infected piglet. Data show that at 25 dpi five HCDR3 bands are prominent in nearly all tissues: pnts 66, 54, 39, 27, and 15. Even the IPP and thymus, primary lymphoid tissues in newborn piglets, have lost their typical unselected Gaussian pattern (compare with Fig. 3, A and B) and resemble other tissues in spectratype. The PBL spectratype was least biased. The examples shown in Figs. 1 and 2 are representative of every PRRSV-infected piglet that we have examined at 10 and 25 dpi.

All PRRSV-infected piglets do not share the same spectratype

Although a common spectratype among different tissues of the same piglets is characteristic of PRRSV infection, the same spectratype is not observed in each animal (Fig. 2B). Although pnt 42 is expanded in 49C and 49B, a major pnt of 27 is pronounced in tissues of 49B and 49D but is absent from 49C (Figs. 1 and 2B). The prominent 42 pnt in 49B and 49C seems replaced in 49D with a 39 pnt (Fig. 2, A and B). Piglet 49D has also uniquely expanded the 66 pnt band, whereas pnt 45 is expanded in 49B. Thus, infection with PRRSV results in a similar spectratype among tissues, but spectratypic patterns are individually specific.

Spectratypes in sham and SIV-infected piglets are not shared among all tissues

A shared tissue-wide HCDR3 spectratype has not been observed in animals infected with other pathogens. Fig. 3 shows that unlike PRRSV-infected piglets, only anatomically adjacent tissues, e.g., colon vs jejunal Peyer’s patches and BAL cells vs lung share a common spectratype in SIV-infected piglets (Fig. 3A). Otherwise, each tissue from the SIV-infected animal displays its own unique spectratype. In both SIV-infected (Fig. 3A) and sham controls (Fig. 3B), primary lymphoid tissue (IPP and thymus) display a near Gaussian polyclonal pattern, whereas this Gaussian pattern is not seen in PRRSV-infected piglets. In sham piglets, anatomically adjacent tissues also share a common spectratype, e.g., colon, jejunum, and MLN. This same spectratype is also shared with BAL cells and PBL but not others. Spl, TON, and lung have their own unique spectratypes. Unlike PRRSV-infected piglets, the spectratype of PBL from sham animals often resembles that of peripheral lymph nodes, e.g., MLN.

VH usage does not significantly differ between PRRS and control piglets

In mouse and humans, B cell SAgs that can potentially expand certain populations target the framework region of certain VH genes (6). Thus, the tissue-wide spectratype among PRRSV-infected piglets that was observed (Figs. 1 and 2) might reflect the selection and expansion of a small number of B cell clones that use a common VH gene. Fig. 4 shows that VH usage by IgM and IgG transcripts from blood PBMCs of PRRSV-infected piglets does not differ from sham-inoculated controls. Because spectratypic analyses indicated that the repertoire of B cells in blood was least representative of those from infected tissues (Figs. 1 and 2), we also compared VH usage among transcripts recovered from blood and those recovered from sites associated with infection. Data show that the pattern observed for BLN and TON was the same as the pattern observed for PBLs (Fig. 5). Thus, data in Figs. 4 and 5 show that B cell hyperplasia in PRRSV-infected piglets is not associated with selective expansion of cells expressing a particular VH gene either in blood or at the site of infection.


Figure 4
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  		FIGURE 4. Average VH usage in nonisotype-switched (IgM) and switched (IgG) PBLs of sham- and PRRSV-inoculated piglets. Data presented are the average proportional VH usage ± SD by IgM-expressing PBLs from sham (A) and PRRSV (B) animals at 0, 7, and 28 dpi. C and D, Parallel data from IgG-expressing PBLs from sham and PRRSV animals. Data from three to seven piglets were averaged for each time point and isotype. Each histogram bar represents the analysis of 270–630 VDJ clones. Statistically significant differences (* and **) for IgG VH usage was age but not treatment related.

 

Figure 5
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  		FIGURE 5. Proportional VH gene usage in VDJ transcripts from B cells from peripheral blood, BLN, and TON of two different PRRSV-infected piglets. A and C, IgM- and IgG-associated VH usage by cells from one infected piglet 28 dpi. B and D, Parallel data from a second infected piglet 28 dpi. On average, 90 clones were analyzed for each isotype from each tissue examined.

 
Repertoire diversification does not occur in PRRSV-infected piglets

In the swine system, 70–80% of the naive preimmune repertoire uses the combination of VHA, VHB, VHC, and VHE in germline configuration (58). Maturation/diversification of this repertoire after Ag encounter results in somatic hypermutation of these major genes and in the use of other minor VH genes (61). This shifts the usage profile (Figs. 4 and 5) to increased usage of "other" so that in a fully diversified repertoire, 80–90% of VH usage would be found in the "other" category (61). Most VH genes in the "other" category are mutated versions of VHA, VHB, VHC, and VHE that no longer hybridize with their gene-specific CDR1- and CDR2-specific probes (see Materials and Methods and Ref. 61). Fig. 4 shows that IgM transcripts from the majority of B cells use the four major VH genes at all three time points, whereas the remainder use "other" VH genes. There was a greater usage of both VHA and "other" by IgG transcripts and proportional usage changed with age. However, the pattern of usage was the same for PRRSV-infected piglets and sham controls. These data for PBMCs and our failure to see any evidence for repertoire diversification when tissues at the site of infection were compared (Fig. 5), indicate that infection with PRRSV does not result in Ab repertoire diversification.

CDR3 sequences from PRRSV-infected piglets have a skewed hydropathicity profile

Because we could not find evidence that B cell expansion in PRRSV-infected piglets results from SAg-like selection of certain VH genes or clones with certain HCDR3 lengths, we shifted our focus to the sequence of HCDR3s in PRRSV-infected piglets. Examples of HCDR3 sequences from piglets in different treatment groups are given in Fig. 6. Boldface underlines denote the sequence of known DH segments and these are identified in a column to the left along with their RF. The complete germline DH segment sequences translated in all RFs are shown at the bottom of the figure. Values for the hydropathicity index (H.I.) of all translated sequences are given. Fig. 6 shows that the H.I. of PRRS HCDR3 sequences was often higher than for HCDR3s from other treatment groups. Thus, we examined >415 HCDR3 sequences of various lengths from PRRSV-infected piglets and controls. Data obtained were used to prepare hydropathicity profiles. Fig. 7 shows that analysis of 92 HCDR3 sequences from three PRRSV-infected piglets yields a hydropathicity profile with major peaks of 0.4 and 0.7 and a mean H.I. 2- to 3-fold higher than in all other groups. Newborns, SIV-infected, and conventional pigs (PIC) all display a major population (60–80% of the HCDR3s) that is neutral to slightly hydrophobic (0.0–0.3), whereas this population is nearly absent in the PRRSV-infected piglet profile. In contrast, a hydrophobic population (H.I. > 0.5) was nearly absent in PIC pigs that had been exposed to many environmental microorganisms, although it was present as a minor subpopulation in newborns. Thus, infection with PRRSV selectively results in the expansion of B cell clones that skews the hydropathicity profile of HCDR3 strongly toward the hydrophobic range.


Figure 6
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  		FIGURE 6. Representative HCDR3 sequences of different length from PRRSV-infected and control animals. Cloned sequences are organized in groups of five and aligned according to the 3' end of FR3 and the beginning of FR4 anchored by the invariant tryptophan. DH segments and their frame (RF) are identified and underlined within the region that also contains N and P additions. The sequence of the often shortened and mutated 5' JH region is also shown. H.I. were calculated over the region between cysteine 104 and the invariant tryptophan that starts FR4. The cysteine and tryptophan residues are in bold. The deduced amino acid sequences and H.I. for DHA and DHB in all RFs is given at the bottom. Brackets indicate RFs containing stop codon that are indicated with dashes.

 

Figure 7
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  		FIGURE 7. H.I. profiles of PRRSV-infected piglets compared with various control piglets. The profile for PRRSV-infected animals (---------) is superimposed on each control. The legend is on the figure and the number of HCDR3 sequences examined is indicated. Major peaks of the profile are labeled regions I, II, and III. The sequences found in each of these regions are analyzed in Table II.

 
The skewed hydropathicity profile of PRRS results from hydrophobic motifs in the HCDR3

We analyzed the most frequently recovered HCDR3 lengths from piglets in all groups and showed that the HCDR3s from PRRSV-infected piglets used DHA three times more frequently than DHB, although this did not differ significantly among treatment groups (Table I). However, RF3 usage in DHA for all lengths of HCDR3 from PRRSV-infected piglets was double or more that seen in other groups (p = 0.0005). RF3 of DHA has a H.I. of 1.35 (Fig. 6) and its usage can explain the skewed hydropathicity profile of PRRSV-infected piglets (Fig. 7). We then related DH and RF usage to three major regions of the hydropathicity profiles indicated in Fig. 7. Analyses of the sequences in regions I, II, and III for each animal treatment group are summarized in Table II. First, this comparison shows that only in PRRSV-infected piglets was there a substantial number of HCDR3 sequences in region I (35 vs 7% for PIC). In all other groups, region III contained the majority of sequences. Second, Table II shows that in region I, 92% of PRRSV-infected piglet sequences used DHA, i.e., all but two, which is significantly greater than in other groups. Neither usage of DHA nor RF3 in region II of PRRSV-infected animals was greater than in other groups (Table II). The skewing of the hydrophobicity profile in PRRS to yield the 0.7 major peak (Fig. 7) is the combined result of a greater proportion of sequences that group to region I in PRRSV-infected piglets and their use of RF3 of DHA. The region II peak in these animals (0.4) is not simply explained by DHA-RF3 usage and this subpopulation is discussed below.


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  		Table II. DH and RF usage of HCDR3 sequences from different regions of hydropathicity profilesa

 
Examination of 24 sequences from hydropathicity profile region I of PRRS indicated that of the 22 that used DHA-RF3, 17 contained the AMVLV motif. Three of the remaining had similar motifs: AMMLV, VLVAM, and MPVAM. The two non-DHA-RF3 sequences in region I had different hydrophobic motifs: IAIVGV and IAILMVL. Thus, the AMVLV motif accounts for 80% of DHA in region I. The AMVLV motif was found in only 10% of sequences in region II, although a number of HCDR3s encoded by RF3 of either DHA or DHB contained related motifs like VLV, MVLVA, IAIVLV, and AVLL. A substantial number of DH segments in the PIC group could not be classified because they were heavily mutated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Polyclonal B cell activation and hypergammaglobulinemia occur in association with various viral infections (7, 8 , and 12) that stimulate type I IFNs that in turn enhance B cell proliferation (66). This hypergammaglobulinemia is often associated with autoimmunity (7, 12, 13) perhaps because of nonspecific proliferation of B cells expressing a preimmune-like repertoire that is known to be polyreactive and to recognize autoantigens (67, 68, 69, 70). Although previous spectratypic analyses show that B cell HCDR3s from PRRSV-infected piglets were a signature for polyclonal activation, certain HCDR3 lengths were pronounced (12). This result differed from the Gaussian profile seen in primary lymphoid tissue (50 ; Thymus and IPP; Fig. 3B) and the non-Gaussian pattern of conventional MLN B cells (Fig. 1A). The unusual B cell phenotype (see below) and our original spectratypic observations (12) prompted this study in which we compared SIV-infected, PRRSV-infected, and sham-inoculated littermates in side-by-side comparisons in an effort to identify unique features of the B cell repertoire in PRRSV-infected piglets.

Data presented in Figs. 4 and 5 indicate that clonal expansion is neither associated with usage of a certain VH gene nor diversification of the Ab repertoire. Lack of a VH-associated SAg effect is not surprising because B cell SAgs are so far known to target FR3 of members of certain VH families, typically VH3 (6, 71). Because all swine VH genes belong to the VH3 family and are >96% homologous (72), a classical B cell SAg would go undetected. In any case, VH gene usage by autoantibodies most often reflects the normal expressed repertoire, not selective VH usage (73).

Because VH usage by virus-infected piglets did not differ from control piglets, we shifted our focus to HCDR3 in which differences in length, when verified by sequence, is a clone marker (B cell clones; Fig. 1A). Spectratypic analysis indicated that for any one PRRSV-infected piglet sampled at the same time, all tissues display a similar pattern suggesting widespread dissemination of the same B cell clones, whereas this was not true for sham controls and those infected with SIV (Figs. 1–3). Because each PRRSV-infected piglet displays a unique spectratype (Fig. 2B), expansion of B cells with BCRs having certain HCDR3 lengths does not define the PRRS spectratype. The predominance of HCDR3 pnt lengths of 36–45 in PRRSV-infected piglets is similar to that in fetal and newborn piglets (50). Thus, selection appears to depend on the probability of encountering B cells expressing the most abundant HCDR3 lengths.

HCDR3 sequence analysis initially revealed that 20–50% of the HCDR3s sequences from PRRSV-infected animals were duplicates, suggesting that only a small number of clones were expanded in PRRS (Table I). However, because duplicates were also recovered frequently in newborn, age-matched sham and SIV-infected piglets but not in PIC pigs (Table I), we believe this phenomenon is age, not PRRSV-related. Duplicate sequences aside, sequence analysis revealed that HCDR3s in PRRSV-infected piglets are hydrophobic compared with all other groups studied (Fig. 7). This result was due to usage of the very hydrophobic region encoded by RF3 of DHA (Table II and Fig. 6). Eighty percent of these hydrophobic HCDR3s (region I, Table II) contain the AMVLV motif, and the remainder in regions I and II have related hydrophobic motifs. The H.I. of the majority of HCDR3s in other piglets are neutral to slightly hydrophobic (H.I. = 0.0 -> 0.3), but this population is conspicuously missing in PRRSV-infected piglets (Fig. 7). The pattern observed for non-PRRSV-infected piglets was similar to that reported in the studies of Schroeder and colleagues (74) in mice and humans. These investigators show that in the mature B cell repertoire, glycine and tyrosine primarily encoded in RF1 are overrepresented and this effect is responsible for the near neutral (0.0–0.3) population (64). Hydrophobic HCDR3s are found in the T1 repertoire and the immature B cells from which they are derived, but this population is selectively lost in mature, circulating B cells (75).

Data presented herein show that so-called "polyclonal B cell activation" in this viral infection is instead selective expansion of B cells with hydrophobic HCDR3s that are normally deselected during development (75) to yield an oligoclonal population with hydrophobic HCDR3s. This biased expansion means that the normally dominant, neutral-charged HCDR3 population seen in newborns and other treatment groups (Fig. 7) is poorly represented in the VH repertoire, consistent with immune dysregulation in this disease. This effect appears to be due to selective proliferation of naive B cells of the preimmune repertoire that do not diversify their repertoire in a T-dependent manner (Figs. 4 and 5). Evidence that these remain naive B cells comes from FCM studies showing that >95% are CD2+, similar to those emerging at birth from marrow whereas only 50% remain CD2+ in SIV-infected littermates (76) (M. Sinkora, K. Kimura, and J. E. Butler, unpublished observations). Failure of PRRSV-infected piglets to develop a normal diversified repertoire with a neutral HCDR3 hydropathicity profile may explain why VN Ab responses are delayed (29), and <1% of the IgG is virus specific in PRRSV-infected isolator piglets (12). This may also explain why PRRSV establishes a persistent infection (40).

Because the hypergammaglobulinemia we reported for PRRS elevates serum IgG, IgM, and IgA in proportion to the levels in fetal serum (60) and Ig aggregates of all three isotypes are deposited (12), we had no reason to suspect that B cell expansion is biased to IgG-secreting cells. It is important to remind readers that class switch combination occurs during fetal life in piglets when they have no exposure to environmental Ag and receive no maternal Ig (60, 76, 77, 78, 79). We considered it more important to compare >415 sequences of one isotype to avoid criticism that our conclusions were drawn from too small of a sample.

We assume that the Igs responsible for the hypergammaglobulinemia in PRRSV-infect piglets result from the B cells with hydrophobic HCDR3s that we report here. The alternative is that the secreted Igs are not derived from the predominant B cells but rather from a minor population within the hydropathicity spectrum (Fig. 7). The autoantibodies in PRRSV-infected piglets are another matter. If they had hydrophobic HCDR3s, it would create an enigma. The best correlation between HCDR3 sequences and autoantibodies are those to dsDNA and these have charged groups like arginines and lysines (80, 81, 82, 83). Ags like dsDNA, Golgi and endothelial cells that are recognized by autoantibodies in PRRSV-infected piglets are unlikely to bind hydrophobic HCDR3s. These autoantibodies might be explained by the small population of B cells with neutral/acidic HCDR3 in PRRSV-infected piglets (Fig. 7). Alternatively, the vascular deposition of IgG, IgM, and IgA that resembles autoantibodies (12) may be deposition of hydrophobic Ig aggregates, not immune complexes. Aggregates of these types have been reported in lactate dehydrogenase-elevating virus (13, 84).

There are few clues to explain B cell selection during PRRSV infection and no published evidence that PRRSV infects B cells (85). Viral epitopes are generally hydrophilic glycoproteins, but amphipathic helices that can be hydrophobic are present in ORF proteins including PRRSV (86). The M protein (ORF6) contains three hydrophobic segments in arteriviruses (87, 88), although the M protein is poorly immunogenic in swine (89). If hydrophobic BCRs recognize an amphipathic helix from PRRSV or segments of the M protein, why doesn’t this trigger Ag processing, sequester T cell help, and result in repertoire diversification? The response is more similar to that of LPS, which causes B cell proliferation and differentiation without regard to BCR specificity. The phenomenon we report occurs in germfree piglets that are free of LPS. B cell selection may depend on which signal pathway is activated (90) and may indeed involve coligation of the BCR and innate immune receptors (91). Perhaps a hydrophobic gene product from PRRSV cross-links hydrophobic BCRs with an innate surface receptor that can signal for proliferation and differentiation in a T-independent fashion.

The reason why the PRRS pandemic has not been eliminated by sterilizing immunity and the connection to the extraordinary B cell events in this syndrome remain speculative. Perhaps PRRSV persists because of the following: 1) the co-lateral damage caused by the T-independent production of autoantibodies, kidney deposition of immune complexes, or aggregated IgG and vasculitis (12) increases susceptibility to other pathogens (47, 49); and/or 2) the low CD4+:CD8+ ratio fails to support Ab repertoire development, isotype switch, and consequently effective VN Abs. The molecular basis of hypergammaglobulinemia and sometimes autoimmunity in some viral infections remains unexplained. Molecular data presented here provide insight into one of these viral infections.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Porkboard Grant 05-174, U.S. Department of Agriculture-Agricultural Research Service Cooperative Agreement 58-3625-4-155, and Grant 523/07/0088 from the Grant Agency of the Czech Republic. Back

2 Address correspondence and reprint requests to Dr. John E. Butler, University of Iowa, Department of Microbiology, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-butler{at}uiowa.edu Back

3 Abbreviations used in this paper: SAg, superantigen; PRRS, porcine reproductive and respiratory syndrome; PRRSV, PRRS virus; HCDR3, H chain third complementary region; dpi, days postinfection; BAL, bronchoalveolar lavage; VN, virus neutralizing; ORF, open reading frame; RF, reading frame; MLN, mesenteric lymph node; FCM, flow cytometric analysis; TON, tonsil; BLN, tracheal-bronchial lymph node; Spl, spleen; IPP, ileal Peyer’s patch; pnt, polynucleotide; BM, bone marrow; PIC, parasite-infected young pig; H.I., hydropathicity index. Back

Received for publication December 20, 2006. Accepted for publication March 7, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ehrlich, R.. 1995. Selective mechanisms utilized by persistent and oncogenic viruses to interfere with antigen producing and presentation. Immunol. Res. 14: 77-97. [Medline]
  2. Hahn, G., R. Jores, E. S. Mocarski. 1998. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc. Natl. Acad. Sci. USA 95: 3937-3942. [Abstract/Free Full Text]
  3. McFadden, G., P. M. Murphy. 2000. Host-related immunomodulators encoded by poxviruses and herpesviruses. Curr. Opin. Microbiol. 3: 371-378. [Medline]
  4. Acha-Orbea, H., D. Finke, A. Attinger, S. Schmid, N. Wehrli, S. Vacheron, I. Xenarios, L. Scarpellino, K. M. Toellner, I. C. MacLennan, S. A. Lutter. 1999. Interplay between mouse mammary tumor virus and the cellular and humoral immune response. Immunol. Rev. 168: 287-303. [Medline]
  5. Tripp, R. A., A. M. Hamilton-Easton, R. D. Cardin, P. Nguyen, F. G. Behm, D. L. Woodland, P. C. Doherty, M. A. Blackman. 1997. Pathogenesis of an infectious mononucleosis-like disease induced by a murine {gamma}-herpes virus; role for a viral superantigen?. J. Exp. Med. 185: 1641-1650. [Abstract/Free Full Text]
  6. Silverman, G. J., J. V. Nayak, A. La Cava. 1997. B cell superantigens: molecular and cellular implications. Int. Rev. Immunol. 14: 259-290. [Medline]
  7. Hunziker, L., M. Recher, A. J. Macpherson, A. Ciurea, S. Freigand, H. Hengartner, R. M. Zinkernagel. 2003. Hypergammaglobulinemia and autoantibody induction mechanisms in viral infection. Nat. Immunol. 4: 343-349. [Medline]
  8. Coutelier, J.-P., G. Coulie, P. Wauters, H. Heremans, J. T. der Logt. 1990. In vivo polyclonal B-lymphocyte activation elicited by murine viruses. J. Virol. 64: 5383-5388. [Abstract/Free Full Text]
  9. Hu, B., C. Even, P. G. Plagemann. 1992. Immune complexes that bind to ELISA plates not coated with antigen in mice infected with lactate dehydrogenase-elevating virus: relationship to IgG2a- and IgG2b-specific polyclonal activation of B cells. Viral Immunol. 5: 27-38. [Medline]
  10. Plagemann, P. G., Q. A. Jones, W. A. Cafruny. 2001. Polyclonal activation of B cells by lactate dehydrogenase-elevating virus is mediated by N-glycans on short ectodomains of the primary envelope glycoprotein. Adv. Exp. Med. Biol. 494: 375-384. [Medline]
  11. Rowland, R. R., C. Even, G. W. Anderson, Z. Chen, B. Hu, P. G. Plagemann. 1994. Neonatal infection of mice with lactate dehydrogenase-elevating virus results in suppression of humoral antiviral immune responses but does not alter the course of viremia or the polyclonal activation of B cells and immune complex formation. J. Gen. Virol. 75: 1071-1081. [Abstract/Free Full Text]
  12. Lemke, C. D., J. S. Haynes, R. Spaete, D. Adolphson, A. Vorwald, K. Lager, J. E. Butler. 2003. Lymphoid hyperplasia resulting in immune dysregulation is caused by PRRSV infection in pigs. J. Immunol. 172: 1916-1925.
  13. Grossmann, A., F. Weiland, E. Weiland. 1989. Autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against Golgi antigens and other subcellular elements. Autoimmunity 2: 201-211. [Medline]
  14. Benfield, D. A., E. Nelson, J. E. Collins, L. Harris, S. M. Goyal, D. Robison, W. T. Christianson, R. B. Morrison, D. Gorcyca, D. Chladek. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J. Vet. Diagn. Invest. 4: 127-133. [Abstract/Free Full Text]
  15. Collins, J. E., D. A. Benfield, W. T. Christianson, L. Harris, J. C. Hennings, D. P. Shaw, S. M. Goyal, S. McCullough, R. B. Morrison, H. S. Joo, et al 1992. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. 4: 117-126. [Abstract/Free Full Text]
  16. Conzelmann, K. K., N. Visser, P. Van Woensel, H. J. Thiel. 1993. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology 193: 329-339. [Medline]
  17. Wensvoort, G., C. Terpstra, J. M. Pol, E. A. ter Laak, M. Bloemraad, E. P. de Kluyver, C. Kragten, L. van Buiten, A. den Besten, F. Wagenaar, et al 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet. Q. 13: 121-130. [Medline]
  18. Zimmerman, J. J., K. J. Yoon, R. W. Wills, S. L. Swenson. 1997. General overview of PRRSV: a perspective from the United States. Vet. Microbiol. 55: 187-196. [Medline]
  19. Halbur, P. G., L. D. Miller, P. S. Paul, X. J. Meng, E. L. Huffman, J. J. Andrews. 1995. Immunohistochemical identification of porcine reproductive and respiratory syndrome virus (PRRSV) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs. Vet. Pathol. 32: 200-204. [Abstract]
  20. Magar, R., R. Larochelle, Y. Robinson, C. Dubuc. 1993. Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold. Can. J. Vet. Res. 57: 300-304. [Medline]
  21. Beyer, J., D. Fichtner, H. Schirrmeier, U. Polster, E. Weiland, H. Wege. 2000. Porcine reproductive and respiratory syndrome virus (PRRSV): kinetics of infection in lymphatic organs and lung. J. Vet. Med. B. 47: 9-25.
  22. Xiao, Z., L. Batista, S. Dee, P. Halbur, M. P. Murtaugh. 2004. The level of virus-specific T cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load. J. Virol. 78: 5923-5933. [Abstract/Free Full Text]
  23. Albina, E.. 1997. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet. Microbiol. 55: 309-316. [Medline]
  24. Neumann, E. J., J. B. Kliebenstein, C. D. Johnson, J. W. Mabry, E. J. Bush, A. H. Seitzinger, A. L. Green, J. J. Zimmerman. 2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J. Am. Vet. Med. Assoc. 227: 385-392. [Medline]
  25. Shimizu, M., S. Yamada, K. Kawashima, S. Ohashi, S. Shimizu, T. Ogawa. 1996. Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus. Vet. Immunol. Immunopathol. 50: 19-27. [Medline]
  26. Olin, M. R., L. Batista, Z. Xiao, S. A. Dee, M. P. Murtaugh, C. C. Pijoan, T. W. Molitor. 2005. Lymphocyte response to porcine reproductive and respiratory syndrome virus. Viral Immunol. 18: 490-499. [Medline]
  27. Samsom, J. N., T. G. de Bruin, J. J. Voermans, J. J. Meulenberg, J. M. Pol, A. T. Bianchi. 2000. Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8+ cells. J. Gen. Virol. 81: 497-505. [Abstract/Free Full Text]
  28. Bautista, E. M., T. W. Molitor. 1997. Cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine. Viral Immunol. 10: 83-94. [Medline]
  29. Lopez, O. J., L. Fuertes, N. Domenech, B. Alvarez, A. Ezquerra, J. Pominquea, J. M. Castro, F. Alovso. 1999. Analysis of cellular immune response in pigs recovered from porcine reproductive and respiratory syndrome infection. Virus Res. 64: 33-42. [Medline]
  30. Lohse, L., J. Nielsen, L. Eriksen. 2004. Temporary CD8+ T-cell depletion in pigs does not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV). Viral Immunol. 17: 594-603. [Medline]
  31. Heinen, P. P., E. A. de Boer-Luijtze, A. T. Bianchi. 2001. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection. J. Gen. Virol. 82: 2697-2707. [Abstract/Free Full Text]
  32. Van Reeth, K., H. Nauwynck. 2000. Proinflammatory cytokines and viral respiratory disease in pigs. Vet. Res. 31: 187-213. [Medline]
  33. Wills, R. W., A. R. Doster, J. A. Galeota, J. H. Sur, F. A. Osorio. 2004. Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus. J. Clin. Microbiol. 41: 58-62.
  34. Labarque, G. G., H. J. Nauwynck, K. Van Reeth, M. B. Pensaert. 2000. Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs. J. Gen. Virol. 81: 1327-1334. [Abstract/Free Full Text]
  35. Loemba, H. D., S. Mounir, H. Mardassi, D. Archambault, S. Dea. 1996. Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus. Arch. Virol. 141: 751-761. [Medline]
  36. Vezina, S. A., H. Loemba, M. Fournier, S. Dea, D. Archambault. 1996. Antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus. Can. J. Vet. Res. 60: 94-99. [Medline]
  37. Chung, H.-K., C. Chase. 2003. Expression of interleukin 10 and interleukin 12 in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). J. Comp. Pathol. 129: 205-212. [Medline]
  38. Royaee, A. R., R. J. Husmann, H. D. Dawson, G. Calzada-Nova, W. M. Dawson, G. Calzada-Nova, W. J. Schnitzlein, F. A. Zuckermann. 2004. Deciphering the involvement of innate immune factors in development of the host immune response to PRRSV vaccination. Vet. Immunol. Immunopathol. 102: 199-216. [Medline]
  39. Suradhat, S., R. Thanawongnuwech. 2003. Up-regulation of interleukin 10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 84: 2755-2760. [Abstract/Free Full Text]
  40. Lopez, O. J., F. A. Osorio. 2004. Role of neutralizing antibodies in PRRSV protective immunity. Vet. Immunol. Immunopathol. 102: 155-163. [Medline]
  41. Gonin, P., B. Pirzadeh, C. A. Gagnon, S. Dea. 1999. Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein. J. Vet. Diagn. Invest. 11: 20-26. [Abstract/Free Full Text]
  42. Weiland, E., M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, F. Weiland. 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. [Medline]
  43. Yang, L., M. L. Frey, K. J. Yoon, J. J. Zimmerman, K. B. Platt. 2000. Categorization of North American porcine reproductive and respiratory syndrome viruses: epitopic profiles of the N, M, GP5 and GP3 proteins and susceptibility to neutralization. Arch. Virol. 145: 1599[Medline]
  44. Yang, L., K.-J. Yoon, Y. Li, J.-H. Lee, J. J. Zimmerman, M. L. Frey, K. M. Harmon, K. B. Platt. 1999. Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates. Arch. Virol. 144: 525-546. [Medline]
  45. Plana-Duran, J., I. Climent, J. Sarraseca, A. Urniza, E. Cortes, C. Vela, J. I. Casal. 1997. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91: involvement of ORF3 and ORF5 proteins in protection. Virus Genes 14: 19-29. [Medline]
  46. Allende, R., W. W. Laegieid, G. F. Kutish, J. A. Galesta, R. W. Wills, F. A. Ososrio. 2000. Porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection. J. Virol. 74: 10834-10837. [Abstract/Free Full Text]
  47. DeBruin, M. G. M., J. N. Samson, J. J. M. Voermans, E. M. A. van Rooij, Y. E. DeVisser, A. T. J. Bianchi. 2000. Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus. Vet. Immunol. Immunopathol. 76: 125-135. [Medline]
  48. Suradhat, S., S. Kesdangsakonwut, W. Sada, S. Buranapraditkum, S. Wongsawang, R. Thanawongnuwech. 2006. Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine. Vaccine 24: 2634-2642. [Medline]
  49. Thacker, E. L. 2006. Lung inflammatory responses. In Mucosal Immunology in Domestic Animals. K. Haverson, B. Charley, and M. Bailey, eds. Vet. Research 37: 469–486.
  50. Butler, J. E., P. Weber, M. Sinkora, J. Sun, S. J. Ford, R. Christenson. 2000. Antibody repertoire development in fetal and neonatal piglets. II. Characterization of heavy chain CDR3 diversity in the developing fetus. J. Immunol. 165: 6999-7011. [Abstract/Free Full Text]
  51. Butler, J. E., J. Sun, P. Weber, D. Francis. 2000. Antibody repertoire development in fetal and neonatal piglets. III. Colonization of the gastrointestinal tracts results in preferential diversification of the pre-immune mucosal B-cell repertoire. Immunology 100: 119-130. [Medline]
  52. Butler, J. E., P. Weber, M. Sinkora, D. Baker, A. Schoenherr, B. Mayer, D. Francis. 2002. Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens. J. Immunol. 169: 6822-6830. [Abstract/Free Full Text]
  53. Sinkora, M., J. E. Butler, Z. Rehakova, J. Sinkora. 2000. Development of g/d thymocytes during ontogeny in pigs: a flow cytometry study. J. Immunol. 165: 1832-1939. [Abstract/Free Full Text]
  54. Vincent, A. L., K. M. Lager, W. Ma, P. Lekcharoensuk, M. R. Gramer, C. Loiacona, J. A. Richt. 2006. Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States. Vet. Microbiol. 118: 212-222. [Medline]
  55. Padlan, E. A.. 1994. Anatomy of the antibody molecule. Mol. Immunol. 31: 169-217. [Medline]
  56. Xu, J. L., M. M. Davis. 2000. Diversity in the CDR3 region of VH is sufficient for most antibody specificities. Immunity 13: 37-45. [Medline]
  57. Butler, J. E., J. Sun, P. Navarro. 1996. The swine immunoglobulin heavy chain locus has a single JH and no identifiable IgD. Int. Immunol. 8: 1897-1904. [Abstract/Free Full Text]
  58. Sun, J., C. Hayward, R. Shinde, R. Christenson, S. P. Ford, J. E. Butler. 1998. Antibody repertoire development in fetal and neonatal piglets. I. Four VH genes account for 80% of VH usage during 84 days of fetal life. J. Immunol. 161: 5070-5078. [Abstract/Free Full Text]
  59. Bonefant, C., I. Vallee, J. Sun, G. Thibault, J. M. Guillaumin, Y. Lebranchu, P. Bardos, J. E. Butler, H. Watier. 2002. Analysis of the human CD4 T lymphocyte proliferation induced by retrovirally-infected porcine lymphoblastoid B cell lines. Xenotransplantation 9: 1-13. [Medline]
  60. Butler, J. E., J. Sun, P. Weber, S. P. Ford, Z. Rehakova, J. Sinkora, K. Lager. 2001. Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus occurs independent of environmental antigen and is only weakly associated with repertoire diversification. J. Immunol. 167: 3239-3249. [Abstract/Free Full Text]
  61. Butler, J. E., P. Weber, N. Wertz. 2006. Antibody repertoire development in fetal and neonatal pigs. XIII. Hybrid VH genes and the preimmune repertoire revisited. J. Immunol. 177: 5459-5470. [Abstract/Free Full Text]
  62. Kyte, J., R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132. [Medline]
  63. Eisenberg, D.. 1984. Three-dimensional structure of membrane and surface proteins. Annu. Rev. Biochem. 53: 595-623. [Medline]
  64. Ivanov, I., J. Link, G. C. Ippolito, H. W. Schroeder, Jr. 2002. Constraints on the hydropathicity and sequence composition of HCDR3 are conserved across evolution. M. Zanetti, Jr, and J. D. Capra, Jr, eds. The Antibodies 43-67. Taylor and Francis, London.
  65. McAleer, J., P. Weber, J. Sun, J. E. Butler. 2005. Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B cell repertoire develops independently from that in blood and mesenteric lymph nodes. Immunology 114: 171-183. [Medline]
  66. Braun, D., I. Caramalho, J. Demengeat. 2002. IFN {alpha}beta enhances BCR-dependent B cell responses. Int. Immunol. 14: 411-419. [Abstract/Free Full Text]
  67. Wardemann, H., S. Yurason, A. Schaefer, J. W. Young, E. Meffre, M. C. Nussenzweig. 2003. Predominant autoantibody production by early human B cell precursors. Science 301: 1374-1377. [Abstract/Free Full Text]
  68. Kearney, J. F., J. Bartels, A. M. Hamilton, A. Lehuen, N. Solvason, M. Vakil. 1992. Development and function of the early B cell repertoire. Int. Rev. Immunol. 8: 247-257. [Medline]
  69. Klinman, D. M., A. M. Steinberg. 1987. Systemic autoimmune disease arises from polyclonal B cells activation. J. Exp. Med. 165: 1755-1760. [Abstract/Free Full Text]
  70. Naparstek, Y., J. Andre-Schwarts, T. Manser, L. Wysocki, L. Breitman, B. D. Stollar, M. Gefter, R. S. Schwartz. 1986. A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus. J. Exp. Med. 164: 614-626. [Abstract/Free Full Text]
  71. Goodyear, C. S., G. J. Silverman. 2005. B cell superantigens: a microbe’s answer to innate-like B cells and natural antibodies. Semin. Immunopathol. 26: 463-484. [Medline]
  72. Sun, J., I. Kacskovics, W. R. Brown, J. E. Butler. 1994. Expressed swine VH genes belong to a small VH gene family homologous to human VH III. J. Immunol. 153: 5618-5627. [Abstract]
  73. Stewart, A. K., C. Huang, A. A. Long, B. D. Stollar, R. S. Schwarts. 1992. VH-gene representation in autoantibodies reflects the normal human B cell repertoire. Immunol. Rev. 128: 101-122. [Medline]
  74. Ippolito, G. C., R. L. Schelonka, M. Zemlin, C. Zemlin, Y. Zhaung, G. L. Gartland, L. Nitschke, J. Pelkonen, K. Rajewsky, H. W. Schroeder, Jr. 2006. Forced enrichment for hydrophobic amino acids in immunoglobulin CDR-H3 impairs splenic B cell development but not antibody production. J. Exp. Med. 203: 1567-1578. [Abstract/Free Full Text]
  75. Schelonka, R. L., J. Tanner, Y. Zhuang, G. L. Gartland, M. Zemlin, H. W. Schroeder, Jr. 2007. Categorical selection of the antibody repertoire in splenic B cells. Eur. J. Immunol. 37: 1010-1021. [Medline]
  76. Butler, J. E., and M. Sinkora. 2007. The isolator piglet: a model for studying the development of adaptive immunity. Immunol. Res. In press.
  77. Butler, J. E.. 1974. Immunoglobulins of the mammary secretions. B. L. Larson, Jr, and V. Smith, Jr, eds. In Lactation: A Comprehensive Treatise Vol. III: 217-255. Academic Press, New York.
  78. Butler, J. E., J. Sun, N. Wertz, M. Sinkora. 2006. Antibody repertoire development in swine. In Antibody Repertoire Development. J. E. Butler, ed. Dev. Comp. Immunol. 30: 199–221.
  79. Butler, J. E., N. Wertz. 2006. Antibody repertoire development in fetal and neonatal piglets. XVII. Fetal IgG subclass transcription revisited with emphasis on new IgG3. J. Immunol. 177: 5480-5489. [Abstract/Free Full Text]
  80. Marion, T. N., M. R. Krishnan, D. D. Desai, N.-T. Jou, D. M. Tillman. 1997. Monoclonal anti-DNA antibodies: structure, specificity and biology. Methods Companion Methods Enzymol. 11: 3-11.
  81. Shlomchik, M. J., A. H. Aucoin, D. S. Pistesky, M. G. Weigert. 1987. Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse. Proc. Natl. Acad. Sci. USA 84: 9150-9154. [Abstract/Free Full Text]
  82. Wloch, M. K., S. H. Clark, G. S. Gilkeson. 1997. Influence of VH CDR3 arginine and light chain pairing on DNA reactivity of a bacterial DNA induced anti-DNA antibody from a BALB/c mouse. J. Immunol. 159: 6083-6090. [Abstract]
  83. Barbas, S. M., H. J. Ditzel, E. M. Salonen, W. P. Yang, G. Silverman, D. R. Barton. 1995. Human autoantibody recognition of DNA. Proc. Natl. Acad. Sci. USA 92: 2529-2533. [Abstract/Free Full Text]
  84. Zitterkopf, N. L., Q. A. Jones, D. S. Bradley, K. Durick, R. R. Rowland, P. G. Plagemann, W. A. Cafruny. 2003. Hydrophobic IgG-containing immune complexes in the plasma of autoimmune MRL/lpr mice, lactate dehydrogenase-elevating virus-infected mice, and pigs: association with transforming growth factor-beta and pH-dependent amplification. Viral Immunol. 16: 511-523. [Medline]
  85. Therrien, D., Y. St-Pierre, S. Dea. 2000. Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells. Arch. Virol. 145: 1099-1116. [Medline]
  86. Oleksiewicz, M. B., A. Botner, P. Norman. 2002. Porcine B cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 83: 1407-1418. [Abstract/Free Full Text]
  87. Chen, Z., L. Kuo, R. R. R. Rowland, C. Even, K. S. Faaberg, P. G. W. Plagemann. 1993. Sequences of 3' end of genome and of 5' end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5' leader and bodies of genomic RNAs. J. Gen. Virol. 74: 643-660. [Abstract/Free Full Text]
  88. DeVries, A. A. F., E. D. Chinside, M. C. Hozinek, P. J. M. Rottier. 1992. Structural proteins of equine arteritis virus. J. Virol. 66: 6294-6303. [Abstract/Free Full Text]
  89. Meulenberg, J. J., A. Petersen den Besten, E. P. DeKluyver, R. J. Moorman, W. M. Schaaper, G. Wensvoort. 1995. Characterization of proteins encoded by ORFs2 to 7 of Lelystad virus. Virology 206: 155-163. [Medline]
  90. Grimaldi, C. M., R. Hicks, B. Diamond. 2005. B cell selection and susceptibility to autoimmunity. J. Immunol. 174: 1775-1781. [Abstract/Free Full Text]
  91. Cancro, M. P., J. F. Kearney. 2004. B cell positive selection: road map to the primary repertoire. J. Immunol. 173: 15-19. [Abstract/Free Full Text]



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