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Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Avenue, St Paul, MN 55108, USA
Correspondence
Michael P. Murtaugh
murta001{at}umn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1S)-subunit of porcine haptoglobin (Hp) by tandem MS sequencing and confirmed by immunoblotting with anti-porcine Hp antibody. Hp is an acute phase haem-binding protein consisting of –β heterodimers that is secreted from the liver in response to stresses, including infection. However, the presence of free 1S-subunit in response to infection is novel and may provide new insights into biochemical processing of Hp and its role in disease pathogenesis, including PRRS. Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It is caused by PRRS virus, a positive-sense, single-stranded RNA virus from the family Arteriviridae. All ages of pigs are susceptible to PRRSV infection, but the clinical manifestations vary with the age and physiological status of the animals. The primary host cell for PRRSV is the macrophage (Pol et al., 1991). Pigs become viraemic within a day of infection. In the early acute stage of PRRS, pro-inflammatory cytokines and interferon are not expressed at significant levels (Albina et al., 1998; Buddaert et al., 1998; Murtaugh et al., 2002; van Reeth & Nauwynck, 2000). After the inapparent innate immune response, systemic dissemination of PRRSV occurs to macrophages and dendritic cells in lymphoid tissues and pigs become persistently infected (Wills et al., 1997; Allende et al., 2000; Xiao et al., 2004).
We hypothesized that biochemical responses to the initial viral infection will help to elucidate mechanisms of PRRSV invasion and establishment of persistent infection. As the first step, we screened serum samples early in the course of infection by mass spectrometry (MS). We discovered a single protein peak in the low molecular mass protein fraction that appeared reproducibly and within 1 day of infection. The peak was identified as free haptoglobin (Hp) alpha 1S (1S)-subunit and was present only in PRRSV-infected pigs. The findings suggest that aberrant processing of Hp may be an early feature of infection. The presence of the 1S-subunit also is an early indicator of PRRSV infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or from uninfected healthy controls. The PRRSV infection status of all the sera was confirmed by quantitative RT-PCR (data not shown).
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). Denatured proteins were removed by centrifugation at 13 200 r.p.m. for 10 min in a microcentrifuge and supernatants were dried in a Savant Speed-Vac (GMI). Proteins were rehydrated with 20 µl autoclaved water and desalted on a C4 resin-loaded Zip-Tip column (Millipore) as described previously (Nelsestuen et al., 2005).
MS.
Samples were run on a Biflex III (Bruker Daltonics) matrix assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometer (MS) operated in linear mode. Laser power was set to 39 % attenuation. Total number of shots collected was 500 per spot. The mass spectrometer was calibrated externally with the +1 and +2 charged states of cytochrome c (Sigma) and spectra were calibrated internally with consistent protein peaks of m/z values of 8330 and 4165. Peaks within a 4–18 kDa mass range were analysed by Bruker XTOF 5.1.1 processing software (Bruker Daltonics). Mass spectra were smoothed (Golay–Savitzky formula by using 15 points) and the background was subtracted (Nelsestuen et al., 2005).
SDS gel electrophoresis.
Acetonitrile-treated serum proteins were resolved by 16.5 % Tris-Tricine SDS-PAGE as described previously (Schagger & von Jagow, 1987). Briefly, samples were loaded on to acrylamide/bis-acrylamide (16.5 % T, 6 % C) mini gels and electrophoresed at 105 V constant in 0.2 M Tris-HCl (pH 8.9) and Tris-Tricine [0.1 M Tris, 0.1 M Tricine (Fisher Scientific) 0.1 % SDS, pH 8.25] as anode and cathode buffers, respectively. Separated protein bands were visualized by staining with Deep Purple total protein stain (Amersham Biosciences) according to the manufacturer's instructions.
Liquid chromatography and tandem MS analysis (LC/MS/MS).
In-gel tryptic digestion was performed on SDS-PAGE-resolved protein bands as described previously (Shevchenko et al., 1996). Digested peptide mixtures were loaded and analysed in an LTQ LC/MS/MS instrument (Thermo Finnigan) (Xie et al., 2005). Peptides were first fractionated by LC in an acetonitrile gradient of 5–95 % and then each fraction was analysed by tandem MS. Spectra were extracted by Sequest version 2.2 software (Thermo Finnigan) and submitted for search against sus_PRRS_con_NCBI_nr database (unknown version, 35324 entries) for trypsin-digested peptides. During the search process, a provision was allowed for mass tolerance of fragment ions and parent ions of 1.0 and 2.0 Da, respectively. Further, the following modifications were specified: dehydrogenation of serine and threonine, deamidation of asparagine and glutamine, oxidation of methionine and phosphorylation of serine, threonine and tyrosine.
Scaffold software (version Scaffold-01_06_15; Proteome Software) was used to validate MS/MS-based peptide and protein identifications. Identification of peptides and proteins were accepted if the matching probability was greater than 50 %.
Two-dimensional electrophoresis.
Reagents were purchased from Fisher Scientific unless noted otherwise. Low molecular mass proteins were extracted in 66.7 % acetonitrile from 500 µl PRRSV-infected and non-infected sera, dried, dissolved in water and precipitated with 4 vols of ice-cold acetone for 15 min. Proteins were collected by microcentrifugation at 10 000 r.p.m. for 10 min at 4 °C and air-dried. Proteins were dissolved in 200 µl rehydration buffer [8 M urea, 20 mM dithiothreitol (DTT), 2 % CHAPS, bromophenol blue, 2 % Ampholyte (Bio-Rad Laboratories)]. First dimensional focusing was performed by using a ReadyStrip-11 cm, pH 3–10 IPG strip (Bio-Rad Laboratories) on a Protean IEF cell apparatus (Bio-Rad Laboratories) according to the manufacturer's instructions. After focusing, the IPG strips were equilibrated for 20 min in equilibration buffer (6 M urea, 1.9 % SDS, 10 mg DTT ml–1 in Tris-Tricine cathode buffer), then placed on a 16.5 % Tris-Tricine Criterion pre-cast gel (Bio-Rad Laboratories). Second dimensional electrophoresis was performed on a Criterion Dodeca cell (Bio-Rad Laboratories) at a constant 105 V. Protein spots were visualized by staining with Deep Purple. In-gel tryptic digestion was performed on SDS-PAGE-resolved protein spots as described previously (Shevchenko et al., 1996). Digested peptide mixtures were loaded and analysed by LC/Electrospray ionization (ESI)/MS/MS (Nelsestuen et al., 2005) on QSTAR Pulsar I-Quadrupole TOF MS (Applied Biosystems) using Analysts QS software (Applied Biosystems). Protein identification was performed by Mascot search engine (Matrix Science) by using the NCBI non-redundant database.
Relative quantification of acetonitrile-extracted porcine serum proteins by iTRAQ labelling.
Acetonitrile-extracted serum proteins were processed, digested with trypsin and labelled with iTRAQ (Applied Biosystems) according to the manufacturer's protocol. The treated proteins were digested with trypsin and each sample was labelled with a different isobaric iTRAQ reagent and subsequently combined in a 1 : 1 ratio. The pooled samples were dried in a Savant Speed-Vac and desalted using Sep Pak C18 cartridges (Waters Corporation). The pooled and labelled samples were flushed through the column at the rate of one drop per second and the column was washed with 3 ml 0.1 % trifluoroacetic acid (TFA). Adsorbed proteins were eluted with 1 ml 80 % acetonitrile in 0.1 % TFA and dried in a Savant Speed-Vac. Dried samples were dissolved and analysed by LC/ESI/MS/MS (Nelsestuen et al., 2005) on a QSTAR Pulsar I-Quadrupole TOF MS by using the Analysts QS software. Protein identification and relative protein expression were determined by Proteinpilot Software (Applied Biosystems) using the non-redundant NCBI protein database.
Western blot analysis.
Acetonitrile-extracted serum samples were resolved in 16.5 % Tris-Tricine SDS-PAGE and transferred to a PVDF membrane (Immobilon-P; Millipore). The membrane was blocked in 5 % non-fat dry milk and 0.1 % Tween-20 (Sigma) in PBS for 12 h. The membrane was probed with rabbit anti-porcine Hp antibody (Immunology Consultants Laboratory) for 1 h, followed by incubation with a 1 : 2000 dilution of horseradish peroxidase-labelled anti-rabbit IgG (Bethyl Laboratories). Enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences) were used according to the manufacturer's instructions to visualize the bands.
Amplification of Hp-1S fragment and cloning.
Primers were designed based on the sequence of Sus scrofa Hp 1S (GenBank accession no. AF492467
[GenBank]
) using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) to amplify the region of 1S of Hp from porcine liver cDNA. The forward and reverse primers were 5'-CGCGGATCCATGGCAGAAACCGGCAATG-3' and 5'-CCGCTCGAGCTGCACCTGATCCACTGG-3', respectively. Underlined bases are specific for Hp. Amplification was performed in a GeneAmp PCR system 2400 (Perkin Elmer) with one cycle of 95 °C for 10 min, 35 cycles at 94 °C for 45 s, 55 °C for 45 s and 72 °C for 45 s and then 72 °C for 10 min. Gel-purified 270 bp products were sequenced (Advanced Genetics Analysis Center, University of Minnesota, USA) and cloned into a modified pET 24b (Novagen) vector (Johnson et al., 2007), which was restriction digested with BamHI and XhoI (Promega) at 37 °C for 2 h. Transformed DH5 cells (Stratagene) were cultured on Luria–Bertani (LB) plates containing kanamycin (30 µg ml–1) at 37 °C overnight and plasmids were extracted from PCR-positive colonies using Qiagen Miniprep kit and sequenced (Advanced Genetic Analysis Center, University of Minnesota, USA).
Expression of recombinant Hp-1S (rHp-1S).
Recombinant plasmids were transformed into competent BL21-RP cells (Stratagene) according to the manufacturer's instructions and cultured on LB plates containing kanamycin (30 µg ml–1) and chloramphenicol (35 µg ml–1). Colonies were initially tested by small-scale test expression by using Ni-NTA spin column (Qiagen) according to the manufacturer's directions. Large-scale protein purification was performed as described previously (Johnson et al., 2007) with the following modifications. IPTG-(1 mM) induced bacterial cells were pelleted by centrifugation at 9000 r.p.m. (Beckman JA-10 rotor) for 10 min at 4 °C, resuspended in 50 ml pellet suspension buffer (50 mM Tris-HCl, 500 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol, 1 mM PMSF) and homogenized (Polytron PT3100; Kinematica) at 4000 r.p.m. for 2 min in the presence of 500 µg DNase, 0.2 mg lysozyme ml–1, 2 % Triton X-100 and 8 U benzonase ml–1. The homogenate was incubated for 10 min at room temperature and centrifuged at 10 000 r.p.m. (Beckman JA-10 rotor) at 4 °C for 15 min. The pellet was resuspended, homogenized and pelleted twice more. Inclusion bodies were resuspended and homogenized in 50 ml solution containing 0.1 % Triton X-100, 10 mM β-mercaptoethanol and 1 mM PMSF. Expressed rHp-1S protein was purified by using a Qiagen Ni-NTA agarose-affinity resin as described previously, except that urea was replaced with 6 M guanidine (Johnson et al., 2007). Purity and molecular mass of purified protein was confirmed by SDS-PAGE and MALDI-TOF. Protein concentration was measured by Bradford assay using BSA standards according to the manufacturer's instructions (Bio-Rad Laboratories).
Anti-rHp-1S antibody production.
The rHp-1S protein was provided to Bethyl Laboratories to produce anti-rHp-1S polyclonal antibodies in goat. The animal was immunized with 200 µg and boosted at 14 days with 100 µg. Serum was obtained before immunization (pre-immune) and high titrated antibody, 1/500 000 by ELISA was obtained 35 days after first immunization.
Purification of anti-rHp-1S antibody.
Five milligrams of rHp-1S was coupled with 1 ml CNBr-activated Sepharose 4B (GE Healthcare) in 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3. Unbound sites were blocked with 0.2 M glycine and the resin was loaded on to a 0.7x10 cm column. To purify anti-rHp-1S, 7 ml goat serum was passed through the column twice and the column was washed with PBS until the OD280 reached the baseline. The resin was washed with 0.1 M glycine, pH 4.7, bound antibody was eluted dropwise with 0.1 M glycine, pH 2.8, into 0.1 M Tris-HCl, pH 9.0. Fractions with the highest optical density values were pooled and dialysed against PBS. Purity of the fractions was confirmed by SDS-PAGE. Protein concentration was measured by Bradford assay using BSA standards (Bio-Rad Laboratories).
Affinity purification of Hp from serum.
Purified anti-rHp-1S antibodies were coupled to CNBr-activated Sepharose 4B (GE Healthcare) as described above. Serum diluted twofold in PBS was passed over the column. Bound protein was washed and eluted as described, and stored at –20 °C in PBS. Concentration was determined by Bradford assay using BSA standards (Bio-Rad Laboratories).
Hp ELISA.
Serum Hp levels were determined by ELISA. Maxisorb plates (Corning) were coated with 200 ng affinity-purified anti-rHp-1S antibodies. Wells were washed and serum was applied at a 1/1000 dilution. Rabbit anti-porcine Hp antibody (1/6000 dilution) was applied for 1 h, the plates were washed three times, and horseradish peroxidase-conjugated goat anti-rabbit IgG (Bethyl Laboratories) was added at a 1/5000 dilution. Colour was developed with 100 µl tetramethylbenzidine peroxidase substrate (KPL) and reactions were terminated after 15 min by adding 100 µl 1 M phosphoric acid. Plates were read at A450 on a Thermo Max Micro plate reader (Molecular Devices). Absorbance values were converted to Hp concentration based on a standard curve run on the same plate by using serially diluted affinity-purified Hp in place of serum.
Software.
ClinProTools version 2.0 (Bruker Daltonics) was used to analyse average mass spectra. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Effect of PRRSV infection on the low molecular mass serum protein profile
Individual mass spectra were obtained from the serum of 25 PRRSV-infected and 34 control porcine samples. A peak with m/z value of 9244±2 consistently appeared only in PRRSV-infected pigs (Fig. 1a and b). No other differences were reproducibly observed between healthy and PRRSV-infected swine serum samples. Also, no differences were observed in spectra <4 or >18 kDa when the above samples were further analysed.
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, P=1.06E-05, Wilcoxon rank sum test). No other peak in the spectra was significantly different between uninfected and infected pigs. The 9244 m/z peak was present in 32 % of animals at day 1 and increased to 92 % by days 5–7. Sensitivity and specificity for detecting PRRSV infection using the 9244 m/z peak were 0.92 and 0.94, respectively (Supplementary Table S1 available in JGV Online). Additional m/z peaks were observed in some samples, but they were not correlated with the PRRSV infection status. The above findings indicate that PRRSV infection caused a characteristic change in the low molecular mass serum protein profile.
To determine if the 9244±2 m/z peak was specific for PRRSV infection, we also analysed the serum protein mass spectra of pigs challenged with other pathogens or antigens. Between two and three of five pigs infected for 5–7 days with Salmonella, Lawsonia intracellularis or swine influenza had the same peak, as did one of four pigs at 2 days after immunization with keyhole limpet haemocyanin. Thus, the peak was observed most reproducibly following PRRSV infection.
Identification of the 9244±2 Da protein
To identify the 9244 Da peak, acetonitrile-extracted serum samples were resolved in SDS-PAGE. Six resulting bands within a 7–13 kDa molecular mass range were excised and subjected to trypsin digestion and analysis by LC/MS/MS. One of the bands present only in PRRSV-infected sera was identified tentatively as Hp-1S (Fig. 2a). Masses of fragmented peptide ions generated from three trypsin-digested peptides matched at 95 % probability with the theoretical mass values of the Hp-1S-subunit peptides (Fig. 2b). The predicted molecular mass of the porcine Hp-1S is 9246 Da, which is consistent with the 9244±2 m/z peak in MALDI-TOF mass spectra of the PRRSV-infected sera. Two-dimensional gel electrophoresis was performed on acetonitrile-extracted sera, followed by LC/MS/MS analysis of the resolved spots, and this confirmed Hp-1S to be the only protein present in the 9244 MW peak (data not shown).
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1S-subunit in porcine sera before and after PRRSV infection1S with 98 % probability. Relative quantitative analysis of signal intensities of reporter ions liberated from the Hp-1S peptide showed that Hp-1S was elevated upon PRRSV infection by 9- to 41-fold compared with a control serum protein, transthyretin (Fig. 2c). No other proteins were found to be altered significantly upon PRRSV infection.
Freely circulating Hp-1S in porcine serum
Hp is a disulphide-bonded tetramer of two -subunits and two β-subunits. To date, freely circulating Hp subunits have not been associated with a host response to infection. Since Hp-1S was detected in the acetonitrile-extracted serum under reducing conditions, we determined if Hp-1S-subunit was freely present in the non-reduced acetonitrile-extracted serum. Thus, SDS-PAGE and Western blot analysis were performed under reducing and non-reducing conditions with anti-porcine Hp antibodies. A band at approximately 9 kDa was observed only in serum from PRRSV-infected pigs, both in reducing and non-reducing conditions (Fig. 2d and e). This result confirms the finding of the iTRAQ experiments showing elevated levels of Hp-1S in PRRSV-infected serum. Further, to determine the kinetics of Hp-1S accumulation, serum was obtained from pigs infected with either of two different PRRSV isolates over a 3–5 week interval. Western blot analysis under non-reducing conditions showed the appearance of free Hp-1S-subunit within 3 days of infection that peaked at 13–21 days (Fig. 2f and g).
Isolation of Hp-1S from PRRSV-infected serum
The preceding experiments suggested that Hp-1S-subunit was freely circulating apart from intact Hp. To further address this possibility, serum from uninfected and PRRSV-infected pigs was passed through a column containing affinity-purified anti-Hp-1S antibodies to isolate molecules containing Hp-1S. SDS-PAGE and MALDI-TOF analysis confirmed the purity and the 12 681 predicted MW of rHp-1S containing myc and his tags (Supplementary Fig. S1 available in JGV Online) used to produce the antibodies. As shown in Fig. 2(h) and (i), serum from a PRRSV-infected pig contained free Hp-1S, whereas serum from an uninfected pig did not, respectively. Boiling and disulphide reduction with DTT increased the amount of Hp-1S about 10-fold (data not shown), indicating that the antibody affinity column isolated both free Hp-1S and intact β Hp. These results, taken together with the findings from acetonitrile-extracted sera (Fig. 2d and e), indicate that Hp-1S is freely circulating in the serum of pigs acutely infected with PRRSV.
Effect of PRRSV infection on serum Hp levels
Total Hp levels were examined in serum of pigs not infected or infected with each of three PRRSV strains that varied in virulence, from moderate (MN30100) to virulent (JA142) and highly virulent (MN184) (Bierk et al., 2001; Johnson et al., 2004). Hp levels were variable, as shown in Fig. 3, but overall there was a significant increase over time (P=0.012, repeated measures ANOVA) and the impact of infection was near significance (P=0.076).
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DISCUSSION |
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1S early in the course of PRRSV infection is interesting and novel. It is not present or present at very low levels in uninfected pigs, but can be detected within 24 h and is present in nearly all infected pigs by 5 days. By contrast, intact Hp is present in the absence of infection and is not significantly or reproducibly increased following PRRSV infection. Hp is primarily synthesized in hepatocytes and secreted into the bloodstream (Hanley et al., 1983; Haugen et al., 1981). Extrahepatic expression of Hp is reported in adipose tissue, uterus and alveolar macrophages in various mammalian species (D'Armiento et al., 1997; Olson et al., 1997; Yang et al., 2000). It is unlikely that PRRSV directly affects Hp biosynthesis since the virus does not infect hepatocytes. Hp mRNA levels in uninfected macrophages is at the margin of quantitative RT-PCR sensitivity and is not affected by PRRSV infection (unpublished data).
The Hp-1S-subunit is translated with the β-subunit as a single polypeptide and is released by cleavage in the endoplasmic reticulum by a complement C1r-like protein (Wicher & Fries, 2004). Humans express three different forms of Hp -subunits, 1S, 1F and 2, that combine with a single β-subunit to generate multimers (Bowman & Kurosky, 1982; Mikkat et al., 2004; Tubbs et al., 2005). Pigs possess one -subunit, which resembles human Hp-1S, with one β-subunit (Ponsuksili et al., 2002). Disulphide bonds are formed to generate the mature tetramer prior to secretion. It is constitutively present in animal serum (Parra et al., 2006). The origin of free Hp-1S is not known. It has been reported previously only in serum of humans with ovarian cancer (Ye et al., 2003). In pigs, its appearance after PRRSV infection is rapid and peaks at 13–21 days of infection, which is later than the typical peak of viraemia at 3–10 days (Chang et al., 2002; Johnson et al., 2004). It is possible that PRRSV pathogenesis indirectly results in hepatocyte damage and release of cleaved Hp-1S.
Expression of Hp is regulated primarily by interleukin (IL)-6 and other inflammatory cytokines, including tumour necrosis factor and IL-1β (Wang et al., 2001). Neither IL-1 nor IL-6 was elevated in porcine serum within the first week of PRRSV infection in two studies (Diaz et al., 2005; Murtaugh and others, unpublished data). A third study found elevated IL-6 levels following infection (Asai et al., 1999). Thus, although PRRSV infection is associated with elevated levels of Hp (Asai et al., 1999; Diaz et al., 2005), these findings suggest that there is not a causal relationship between PRRSV infection and Hp levels. Hp is associated with weight gain in growing pigs (Eurell et al., 1992). Substantial variation has been reported in Hp levels in swine, suggesting that its regulation may be influenced by a variety of factors (Petersen et al., 2001; Piñeiro et al., 2007). Even if PRRSV infection was associated with an elevation in serum Hp, the quantitative increase would be small, about two to threefold, which is well below the 10-fold range for an acute phase Hp response in pigs (Petersen et al., 2004). It also is less than the 9- to 41-fold increase in free Hp-1S observed here.
Hp is a classic acute phase protein, although its precise functions are not fully understood. It binds to CD11b/CD18, an integrin receptor that is expressed on monocytes, granulocytes, subsets of CD8+ T cells and natural killer cells (El Ghmati et al., 1996). Hp also regulates the release of cytokines by type 2 helper T cells (Arredouani et al., 2003) and binds to B cells via the lectin CD22 receptor (Hanasaki et al., 1995). Hp is also a potent antioxidant (Tseng et al., 2004). Its most important function in the acute phase response to infection is binding and sequestering free haemoglobin (Hb) in the circulation, thus reducing free iron availability to microbial pathogens (reviewed by Petersen et al., 2004). The complex facilitates scavenging of Fe3+ via CD163 receptors on macrophages (Kristiansen et al., 2001). The Hp–Hb complex interacts with CD163 exclusively through the β-subunit of Hp and the -globin of Hb, without direct involvement of the -subunit of Hp (Kristiansen et al., 2001). These features make Hp an unlikely host defence molecule in PRRSV infection since the infection is not haemolytic and the virus has no requirement for extracellular iron.
Recently, CD163 was identified as a necessary receptor for PRRSV infection (Calvert et al., 2007). This striking coincidence may link a physiological acute phase response with an infectious disease process. At this point a direct connection between PRRSV infection and Hp–Hb binding is inapparent since free Hp-1S provides little insight into viral pathogenesis or immunity. Nevertheless, the presence of free Hp-1S in the first day is the earliest evidence of a physiological and biochemical consequence of PRRSV infection, and provides an early warning biomarker for serological detection of infection.
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ACKNOWLEDGEMENTS |
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Received 6 May 2008;
accepted 2 July 2008.
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