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Recombinant protein-based ELISA for detection and differentiation of antibodies against avian reovirus in vaccinated and non-vaccinated chickens
Journal of Virological Methods 165 (2010) 108–111
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Recombinant protein-based ELISA for detection and differentiation of antibodies against avian reovirus in vaccinated and non-vaccinated chickens Zhixun Xie a,∗ , Chunxiang Qin a , Liji Xie a , Jiabo Liu a , Yaoshan Pang a , Xianwen Deng a , Zhiqin Xie a , Mazhar I. Khan b,∗∗,1 a b
Department of Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai Road, Nanning, Guangxi 530001, China Department of Pathobiology & Veterinary Science, University of Connecticut, 61 North Eagleville Road Storrs, CT 06269-3089, USA
a b s t r a c t Article history: Received 7 August 2009 Received in revised form 8 December 2009 Accepted 10 December 2009 Available online 21 December 2009 Keywords: ARV Nonstructural proteins Indirect ELISA
Two nonstructural genes, NS and P17, of avian reovirus (ARV) were cloned into the expression plasmid vector PGEX4T-1. Expressed proteins for NS and P17 of avian reovirus were purified and used as antigens. Three indirect NS enzyme-linked immunosorbent assays (ELISAs), NS-ELISA, P17-ELISA and NS-P17ELISA were optimized and used as specific tests. Serum samples from reovirus-infected and vaccinated SPF chickens were tested with the three ELISAs and an agar gel precipitin (AGP) method. ELISAs specific for NS, P17 and NS-P17 were able to detect specific antibodies for avian reovirus in 88.9%, 61.1%, and 88.9% in infected samples, respectively, whereas the AGP detected 55.6% of the infected samples. The detection rates of ELISA specific antibodies for NS, P17 and NS-P17 on sera of vaccinated chickens were 6.7%, 0% and 6.7%. However, in comparison the AGP method detected 60.6% of antibodies in serum samples from vaccinated chickens. The results showed that the use of ELISAs specific for the nonstructural proteins might be able to distinguish between reovirus vaccinated and infected chickens. Further studies are in progress to validate these recombinant protein-based ELISAs under field conditions. © 2009 Elsevier B.V. All rights reserved.
Avian reovirus (ARV) is an important cause of diseases in poultry. Reovirus-induced arthritis, chronic respiratory disease, and malabsorption syndrome (Fahey and Crawley, 1954; Hieronymus et al., 1983; Kibenege and Wilcox, 1983) cause significant economic losses. Many laboratory methods have been developed for the detection of antibodies against ARV, including serum neutralization (Lee et al., 1992; Wickramasinghe et al., 1993), immunodiffusion (Meanger et al., 1995), immunoblot assay (Ide and Dewitt, 1979), and immunofluorescence (Ide, 1982). Although these methods are useful for the detection of ARV infection, most of them are laborious and time-consuming. On the other hand, enzyme-linked immunosorbant assays (ELISAs) have a high level of sensitivity and reproducibility and allow for automation. They are the method of choice for screening a large number of serum samples. Several ELISAs using whole virus or a recombinant ARV protein have been described (Shien et al., 2000; Liu et al., 2000, 2002; Slaght et al., 1978, 1979; Islam and Jones, 1988; Roberson and Wilcox, 2000; Chen et al., 2004). None of these
∗ Corresponding author. Tel.: +86 771 3120371. ∗∗ Corresponding author. Tel.: +1 860 486 0228. E-mail addresses: [email protected] (Z. Xie), [email protected] (M.I. Khan). 1 Visiting Scholar at the Guangxi Veterinary Research Institute. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.12.008
ELISAs distinguishes between ARV-vaccinated and infected serum samples. The nonstructural protein NS encoded by genome segment S4, is one of the four nonstructural proteins in ARV and contains three epitopes that are highly conserved among virus strains (Hou et al., 2001; Huang et al., 2005; Varela and Benavente, 1994; Xie et al., 2007a). The nonstructural protein p17 that is associated with membranes is conserved in every avian reovirus strain and works as a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. It has been demonstrated that p17 is also capable of causing cell growth retardation (Costas et al., 2005; Liu et al., 2005; Qin et al., 2006). ELISAs specific for nonstructural proteins of hepatitis C and influenza viruses have been described that distinguish vaccinated from infected clinical samples (Inoue et al., 1992; BirchMachin et al., 1997; Ozaki et al., 2001). Methods for differentiating vaccine and wild type ARV would be useful for diagnosis and for studying the epidemiology of ARV infection in poultry. Recombinant nonstructural protein-based ELISAs were developed and are described below. The ARV strain S1133 (University of Connecticut, USA) was used for the development of recombinant nonstructural protein-based ELISAs. The virus was propagated in chicken embryo fibroblasts (CEF) at 37 ◦ C for 36–48 h (Wu et al., 1994). The culture was treated by freezing and thawing three times, and thawed culture fluid was clarified by centrifugation at 10,000 × g for 15 min. Virus titration was done in microtiter plates as described by Lee
Z. Xie et al. / Journal of Virological Methods 165 (2010) 108–111
et al. (1992), and the titer was calculated using the Reed and Munch method (1938). Virus with a titer of 106.2 TCID50/mL was inactivated with 0.1% formaldehyde at 37 ◦ C overnight and then emulsified in mineral oil and used as a vaccine. RNA extractions from ARV strain S1133 were carried out using Trizol, according to the manufacturer’s protocol (Trizol, Invitrogen, Carlsbad, CA, USA). To amplify the full-length nonstructural encoding genes of the NS and P17 proteins, purified genomic dsRNA from ARV was used to generate cDNA by reverse transcription and polymerase chain reaction (RT-PCR). The PCR primers XZNS6: 5-GCGAATTCGCCATGGACAACACCGTGC-3 and XZNS9: 5-GCCTCGAGCTACGCCATCCTAGCTGG-3 to amplify the 1107 bp of the NS gene, and the primers P17-1: 5-TGAATTCAGCACAATGCAATGGCTCCGC-3 and P17-2: 5-GCTCGAGTTGGTCAGTCGTTCATA-3 to amplify the 457 bp of the P17 gene were designed and synthesized according to the NS (U95952) and P17 (AF330703) gene sequences of ARV S1133 available in GenBank. PCR products derived from the NS and P17-encoding genes of ARV S1133 were digested with enzyme and then ligated into the corresponding sites of a PGEX4T-1 expression vector. Recombinant plasmids were transformed in Escherichia coli competent cells (DH5␣). Positive colonies pGEX4T-1-NS and pGEX4T-1-P17 identified by PCR and double digestion were sequenced (TAKARA, Dalian, China). The procedures for NS and P17 expression in DH5␣ E. coli have been described previously (Xie et al., 2008, 2007b). Briefly, after induction for 4 h with isopropylthin-B-d-galactoside (IPTG) at a final concentration of 0.4 mM in culture medium, pGEX4T-1-NS and pGEX-4T-1-P17 were induced. The NS and P17 protein were isolated and purified as described (Xie et al., 2008, 2007a), and the recombinant protein concentration was determined according to the method of Lowry et al. (1951). Purified NS and P17 fusion proteins were mixed with an equal volume of Laemmeli sample buffer, boiled for 5 min, and separated by SDS-PAGE using a 10% gel, in a BioRad MiniProtein II electrophoresis unit. After electrophoresis, the fusion proteins were transferred to nitrocellulose membranes by the method of Towbin et al. (1979). The blots were incubated with a 1:200 dilution of chicken anti-ARV S1133 hyper-immune serum (China Institute of Veterinary Drug Control) for 2 h at room temperature. To determine the optimum working concentration of ARV antigens in NS-ELISA, P17-ELISA and NS-P17-ELISA, checkerboard titrations were carried out between the NS, P17, NS-P17 proteins and positive and negative sera (China Institute of Veterinary Drug Control). One hundred microlitres of the purified NS, P17 and NS-P17 proteins in 0.05 M carbonate buffer, pH 9.6, was coated onto the wells of an ELISA plate and tested with twofold serially diluted ARV positive and negative sera, respectively. Horseradish peroxidase (HRP)-labeled goat anti-chicken IgG conjugate was used at a dilution of 1:5000. Purified NS and P17 proteins were used at twofold dilutions (2.33–18.6 g and 2.87–23 g separately) in 0.05 M carbonate buffer, pH 9.6. NS and P17 proteins were mixed and used at a twofold dilution. One hundred microlitre of antigen was added to each well of the ELISA plate. Antigen was coated onto the wells by incubation at 4 ◦ C overnight. The wells were washed three times with phosphate-buffered saline (PBS), pH 7.4 and 0.05% Tween 20 washing buffer. Then 50 L of blocking buffer (PBS, 0.05% Tween 20, and 2.5% bovine serum albumin) were added to each well and incubated for 1 h at 37 ◦ C to saturate all unbound sites. Coated plates were washed three times with PBS and 0.05% Tween 20. Chicken sera diluted at twofold dilutions (from 1:100 to 1:800) using dilution buffer PBS and 0.1% Tween 20, were added at 100 L/well and incubated for 1 h at 37 ◦ C. After incubation and three washing cycles as described above, one hundred microlitre per well of a 1:5000 dilution of horseradish peroxidase (HRP)-labeled goat anti-chicken IgG was added, and the plates
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were incubated for 1 h at 37 ◦ C and then washed three times with washing buffer. A volume of 100 L substrate solution containing 0.04% orthophenylenediamine in 0.05 M phosphate–citrate buffer, pH 5.0 and 0.04% H2 O2 was added. The reaction was carried out for 30 min at room temperature. Color development was stopped after 10 min by adding 50 L of 2.5 M H2 SO4 to each well. The absorbance was read at 450 nm using a plate reader. Duplicate positive and negative control chicken sera were included in each plate. Fifty ARV negative serum samples (China Institute of Veterinary Drug Control) were tested at 450 nm to calculate the average (X) and Stdev (SD) of the OD450 nm values. The cut-off value was calculated according to the formula: cut-off value = X + 3SD. Forty-eight 1-week-old specific pathogenic free (SPF) chickens were divided into 3 groups. Fifteen chickens in group I were injected intramuscularly with inactivated vaccine of 104 TCID50/bird at 2 and 4 weeks old. In group II, 18 2-week-old chickens received ARV live virus at 104 TCID50/bird by eye drop. Fifteen chickens in group III were used as negative controls. Serum samples were collected at 7 weeks of age. Thirty-three sera from chickens infected or vaccinated with AVR were tested by NS-ELISA, P17-ELISA and NS-P17-ELISA separately. Using the whole virus antigen of ARV S1133, thirty-three sera from infected chickens and 33 from vaccinated chickens were tested by agar gel precipitation (AGP) as described (Ide and Dewitt, 1979). For specificity of ELISAs, serum samples from birds with NDV, AIV and IBV were tested by NS-ELISA, P17-ELISA and NS-P17ELISA separately. Statistical analyses of the data were performed by Kruskal–Wallis one-way analysis of variance with the Dunn’s post hoc with SigmaStat Version 3.0. The recombinant plasmids pGEX-4T-1-NS and pGEX-4T-1P17 were identified by PCR (data not shown). It was confirmed by DNA sequencing that the acquired recombinant plasmid contained complete NS and P17 genes, and the pGEX-4T-1-NS and pGEX-4T-1-P17 recombinant plasmids were successfully constructed. Analysis of the purified protein by SDS-PAGE revealed the expressed NS and P17 fusion proteins, with an approximate molecular mass of 66.2 kDa and 42.4 kDa, respectively. These were consistent with the expected size of the fusion proteins (Figs. 1A and B and 2), and the concentrations were 28 mg/mL and 46 mg/mL, respectively. Conditions for NS-ELISA, P17-ELISA and NS-P17-ELISA were standardized by using checkerboard titrations. The results show that, when the concentrations of NS, P17 and NS-P17 were 9.3 g/mL, 11.5 g/mL and 9.3 g/mL, respectively, at serum dilutions of 1:200, the NS-ELISA, P17-ELISA and NS-P17-ELISA gave a clear difference in absorbance values between positive and negative sera. According to 50 ARV negative serum samples, the cut-off value of NS-ELISA was 0.630, P17ELISA was 0.560 and NS-P17-ELISA was 0.640, respectively. NS-ELISA, P17-ELISA and NS-P17-ELISA differentiated ARV positive and negative antibodies in experimentally infected and vaccinated chickens (Tables 1 and 2). The NS-ELISA, P17-ELISA and NS-P17-ELISA were able to detect 88.9%, 61.1%, and 88.9%, respectively of ARV-infected serum samples, whereas the AGP test detected 55.6%. Detection rates on serum samples from the vaccinated SPF chickens were 6.7%, 0% and 6.7%, respectively for these nonstructural proteins. The AGP method detected 60.6% of samples positive for antibodies from ARV-vaccinated chickens. The NSELISA, P17-ELISA and NS-P17-ELISA showed no positive reactions with serum samples from birds with NDV, AIV and IBV, which indicated that these indirect ELISAs had good specificity for the detection of ARV antibody. The nonstructural NS and P17 proteins were expressed successfully and the purified proteins were used as ELISA antigens to detect antibodies against ARV. The purified antigens demonstrated good specificity in ELISAs. These antigens reacted specifically with
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Z. Xie et al. / Journal of Virological Methods 165 (2010) 108–111
Table 1 The OD450 values of individual serum samples. ELISA
NS
P17
NS-P17
NS
P17
NS-P17
NS
P17
NS-P17
Sera collected from chickens infected with ARV
0.781 1.279 0.744 1.086 0.606 0.711
0.686 0.518 0.871 0.795 0.462 0.669
0.808 1.057 0.771 1.068 0.629 0.779
1.167 0.822 0.945 0.674 0.752 0.732
0.947 0.689 1.013 0.483 0.688 0.512
1.076 0.842 1.075 0.726 0.843 0.820
1.395 0.574 1.099 0.899 0.720 1.198
1.049 0.483 1.158 0.427 0.548 0.909
1.118 0.612 1.216 0.917 0.683 1.105
Sera collected from chickens vaccinated with ARV inactive vaccine
0.431 0.617 0.294 0.379 0.470
0.382 0.418 0.521 0.373 0.358
0.298 0.437 0.518 0.272 0.365
0.428 0.223 0.697 0.582 0.285
0.307 0.311 0.491 0.468 0.344
0.353 0.341 0.693 0.265 0.525
0.306 0.346 0.317 0.407 0.516
0.490 0.252 0.385 0.508 0.386
0.326 0.389 0.354 0.476 0.622
Table 2 Detection rates positive for ARV in sera from chickens using ELISA and AGP. Detection method NS-ELISA P17-ELISA NS-P17-ELISA AGP
Infected with ARV *
88.9% 61.1% 88.9%* 55.6%
Vaccinated with ARV 6.7% 0% 6.7% 60.6%
* Detection with NS-ELISA and NS-P17-ELISA was significantly (P ≤ 0.05) different from P17-ELISA and AGP groups.
Fig. 1. Western blot analysis of ARV positive antiserum specific to the expressed NS (A) and P17 (B) fusion proteins. (A) Lane M, protein marker; lane 1, purified NS protein; lane 2, PGEX4T-1 induce at 4 h. (B) Lane M, protein marker; lane 1, PGEX4T-1 induce at 4 h; lane 2, purified P17 protein.
their respective serum samples, did not cross-react with any heterologous sera and exhibited negative reactivity with antisera against Newcastle disease virus, avian influenza virus and infectious bronchitis virus. Recombinant protein-based ELISA is less sensitive but more specific than the whole virus lysate-based ELISA. A previous report (Liu et al., 2002) demonstrated that the combined proteins-based ELISA, compared with the single protein-based
ELISA or whole virus lysate ELISA, could greatly reduce non-specific binding reactions and had a higher correlation with serum neutralization assays. The current results suggest that NS-ELISA, P17-ELISA and NS-P17-ELISA were able to detect antibody in serum samples from the ARV-infected chickens. Using NS and P17 proteins together as ELISA antigens did not increase the sensitivity of the assay. However, NS-ELISA, P17-ELISA and NS-P17-ELISA each was more sensitive than the AGP test (Table 2). The nonstructural proteins are expressed in large amounts in virus-infected cells, but they have not been detected in virions (Krug and Etkind, 1973). It has been known that ELISAs specific for nonstructural proteins of hepatitis C and equine influenza viruses might be able to distinguish vaccination from infection (Inoue et al., 1992; Birch-Machin et al., 1997; Ozaki et al., 2001). In our study, positive detection rates for NS-ELISA, P17-ELISA and NS-P17ELISA were high on the serum samples of infected chickens and vaccinated chickens (Table 2). Thus using NS and P17 as antigens in ELISA can differentiate between sera from vaccinated and infected chickens. The NS-ELISA, P17-ELISA and NS-P17-ELISA were found to be more sensitive than the AGP test (Table 2). This study showed that NS-ELISA was more sensitive than the P17ELISA. A plausible reason may be that the NS protein carries more broadly specific epitopes than the P17 protein. Further testing on a large numbers of serum samples from field cases will be required to confirm these findings. Serum samples from chickens infected with ARV were positive with ELISAs between 61.11% and 88.89%, however not all of the samples were positive. This may be because the antibody level was insufficient for detection by ELISA. The positive rate for 15 serum samples from vaccinated chickens was 0–6.7%. Not all the samples were negative. That may be because the vaccines were not purified, or because booster vaccinating makes more nonstructural proteins. Further studies will be carried out to elucidate these phenomena. In conclusion, the NS-ELISA and NS-P17-ELISA have advantages over the conventional ELISA, with less non-specific binding reactions and stable sensitivity. These could be used to distinguish vaccinated from infected chickens. Acknowledgements
Fig. 2. Purified avian reovirus nonstructural recombinant ARV proteins. Lane 1, P17; lane 2, NS; lane 3, protein marker.
The authors thank Dr. Herbert Van Kruriningen for critical reading of the manuscript. This study was supported by the National
Z. Xie et al. / Journal of Virological Methods 165 (2010) 108–111
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Recombinant protein-based ELISA for detection and differentiation of antibodies against avian reovirus in vaccinated and non-vaccinated chickens Zhixun Xie a,∗ , Chunxiang Qin a , Liji Xie a , Jiabo Liu a , Yaoshan Pang a , Xianwen Deng a , Zhiqin Xie a , Mazhar I. Khan b,∗∗,1 a b
Department of Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai Road, Nanning, Guangxi 530001, China Department of Pathobiology & Veterinary Science, University of Connecticut, 61 North Eagleville Road Storrs, CT 06269-3089, USA
a b s t r a c t Article history: Received 7 August 2009 Received in revised form 8 December 2009 Accepted 10 December 2009 Available online 21 December 2009 Keywords: ARV Nonstructural proteins Indirect ELISA
Two nonstructural genes, NS and P17, of avian reovirus (ARV) were cloned into the expression plasmid vector PGEX4T-1. Expressed proteins for NS and P17 of avian reovirus were purified and used as antigens. Three indirect NS enzyme-linked immunosorbent assays (ELISAs), NS-ELISA, P17-ELISA and NS-P17ELISA were optimized and used as specific tests. Serum samples from reovirus-infected and vaccinated SPF chickens were tested with the three ELISAs and an agar gel precipitin (AGP) method. ELISAs specific for NS, P17 and NS-P17 were able to detect specific antibodies for avian reovirus in 88.9%, 61.1%, and 88.9% in infected samples, respectively, whereas the AGP detected 55.6% of the infected samples. The detection rates of ELISA specific antibodies for NS, P17 and NS-P17 on sera of vaccinated chickens were 6.7%, 0% and 6.7%. However, in comparison the AGP method detected 60.6% of antibodies in serum samples from vaccinated chickens. The results showed that the use of ELISAs specific for the nonstructural proteins might be able to distinguish between reovirus vaccinated and infected chickens. Further studies are in progress to validate these recombinant protein-based ELISAs under field conditions. © 2009 Elsevier B.V. All rights reserved.
Avian reovirus (ARV) is an important cause of diseases in poultry. Reovirus-induced arthritis, chronic respiratory disease, and malabsorption syndrome (Fahey and Crawley, 1954; Hieronymus et al., 1983; Kibenege and Wilcox, 1983) cause significant economic losses. Many laboratory methods have been developed for the detection of antibodies against ARV, including serum neutralization (Lee et al., 1992; Wickramasinghe et al., 1993), immunodiffusion (Meanger et al., 1995), immunoblot assay (Ide and Dewitt, 1979), and immunofluorescence (Ide, 1982). Although these methods are useful for the detection of ARV infection, most of them are laborious and time-consuming. On the other hand, enzyme-linked immunosorbant assays (ELISAs) have a high level of sensitivity and reproducibility and allow for automation. They are the method of choice for screening a large number of serum samples. Several ELISAs using whole virus or a recombinant ARV protein have been described (Shien et al., 2000; Liu et al., 2000, 2002; Slaght et al., 1978, 1979; Islam and Jones, 1988; Roberson and Wilcox, 2000; Chen et al., 2004). None of these
∗ Corresponding author. Tel.: +86 771 3120371. ∗∗ Corresponding author. Tel.: +1 860 486 0228. E-mail addresses: [email protected] (Z. Xie), [email protected] (M.I. Khan). 1 Visiting Scholar at the Guangxi Veterinary Research Institute. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.12.008
ELISAs distinguishes between ARV-vaccinated and infected serum samples. The nonstructural protein NS encoded by genome segment S4, is one of the four nonstructural proteins in ARV and contains three epitopes that are highly conserved among virus strains (Hou et al., 2001; Huang et al., 2005; Varela and Benavente, 1994; Xie et al., 2007a). The nonstructural protein p17 that is associated with membranes is conserved in every avian reovirus strain and works as a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. It has been demonstrated that p17 is also capable of causing cell growth retardation (Costas et al., 2005; Liu et al., 2005; Qin et al., 2006). ELISAs specific for nonstructural proteins of hepatitis C and influenza viruses have been described that distinguish vaccinated from infected clinical samples (Inoue et al., 1992; BirchMachin et al., 1997; Ozaki et al., 2001). Methods for differentiating vaccine and wild type ARV would be useful for diagnosis and for studying the epidemiology of ARV infection in poultry. Recombinant nonstructural protein-based ELISAs were developed and are described below. The ARV strain S1133 (University of Connecticut, USA) was used for the development of recombinant nonstructural protein-based ELISAs. The virus was propagated in chicken embryo fibroblasts (CEF) at 37 ◦ C for 36–48 h (Wu et al., 1994). The culture was treated by freezing and thawing three times, and thawed culture fluid was clarified by centrifugation at 10,000 × g for 15 min. Virus titration was done in microtiter plates as described by Lee
Z. Xie et al. / Journal of Virological Methods 165 (2010) 108–111
et al. (1992), and the titer was calculated using the Reed and Munch method (1938). Virus with a titer of 106.2 TCID50/mL was inactivated with 0.1% formaldehyde at 37 ◦ C overnight and then emulsified in mineral oil and used as a vaccine. RNA extractions from ARV strain S1133 were carried out using Trizol, according to the manufacturer’s protocol (Trizol, Invitrogen, Carlsbad, CA, USA). To amplify the full-length nonstructural encoding genes of the NS and P17 proteins, purified genomic dsRNA from ARV was used to generate cDNA by reverse transcription and polymerase chain reaction (RT-PCR). The PCR primers XZNS6: 5-GCGAATTCGCCATGGACAACACCGTGC-3 and XZNS9: 5-GCCTCGAGCTACGCCATCCTAGCTGG-3 to amplify the 1107 bp of the NS gene, and the primers P17-1: 5-TGAATTCAGCACAATGCAATGGCTCCGC-3 and P17-2: 5-GCTCGAGTTGGTCAGTCGTTCATA-3 to amplify the 457 bp of the P17 gene were designed and synthesized according to the NS (U95952) and P17 (AF330703) gene sequences of ARV S1133 available in GenBank. PCR products derived from the NS and P17-encoding genes of ARV S1133 were digested with enzyme and then ligated into the corresponding sites of a PGEX4T-1 expression vector. Recombinant plasmids were transformed in Escherichia coli competent cells (DH5␣). Positive colonies pGEX4T-1-NS and pGEX4T-1-P17 identified by PCR and double digestion were sequenced (TAKARA, Dalian, China). The procedures for NS and P17 expression in DH5␣ E. coli have been described previously (Xie et al., 2008, 2007b). Briefly, after induction for 4 h with isopropylthin-B-d-galactoside (IPTG) at a final concentration of 0.4 mM in culture medium, pGEX4T-1-NS and pGEX-4T-1-P17 were induced. The NS and P17 protein were isolated and purified as described (Xie et al., 2008, 2007a), and the recombinant protein concentration was determined according to the method of Lowry et al. (1951). Purified NS and P17 fusion proteins were mixed with an equal volume of Laemmeli sample buffer, boiled for 5 min, and separated by SDS-PAGE using a 10% gel, in a BioRad MiniProtein II electrophoresis unit. After electrophoresis, the fusion proteins were transferred to nitrocellulose membranes by the method of Towbin et al. (1979). The blots were incubated with a 1:200 dilution of chicken anti-ARV S1133 hyper-immune serum (China Institute of Veterinary Drug Control) for 2 h at room temperature. To determine the optimum working concentration of ARV antigens in NS-ELISA, P17-ELISA and NS-P17-ELISA, checkerboard titrations were carried out between the NS, P17, NS-P17 proteins and positive and negative sera (China Institute of Veterinary Drug Control). One hundred microlitres of the purified NS, P17 and NS-P17 proteins in 0.05 M carbonate buffer, pH 9.6, was coated onto the wells of an ELISA plate and tested with twofold serially diluted ARV positive and negative sera, respectively. Horseradish peroxidase (HRP)-labeled goat anti-chicken IgG conjugate was used at a dilution of 1:5000. Purified NS and P17 proteins were used at twofold dilutions (2.33–18.6 g and 2.87–23 g separately) in 0.05 M carbonate buffer, pH 9.6. NS and P17 proteins were mixed and used at a twofold dilution. One hundred microlitre of antigen was added to each well of the ELISA plate. Antigen was coated onto the wells by incubation at 4 ◦ C overnight. The wells were washed three times with phosphate-buffered saline (PBS), pH 7.4 and 0.05% Tween 20 washing buffer. Then 50 L of blocking buffer (PBS, 0.05% Tween 20, and 2.5% bovine serum albumin) were added to each well and incubated for 1 h at 37 ◦ C to saturate all unbound sites. Coated plates were washed three times with PBS and 0.05% Tween 20. Chicken sera diluted at twofold dilutions (from 1:100 to 1:800) using dilution buffer PBS and 0.1% Tween 20, were added at 100 L/well and incubated for 1 h at 37 ◦ C. After incubation and three washing cycles as described above, one hundred microlitre per well of a 1:5000 dilution of horseradish peroxidase (HRP)-labeled goat anti-chicken IgG was added, and the plates
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were incubated for 1 h at 37 ◦ C and then washed three times with washing buffer. A volume of 100 L substrate solution containing 0.04% orthophenylenediamine in 0.05 M phosphate–citrate buffer, pH 5.0 and 0.04% H2 O2 was added. The reaction was carried out for 30 min at room temperature. Color development was stopped after 10 min by adding 50 L of 2.5 M H2 SO4 to each well. The absorbance was read at 450 nm using a plate reader. Duplicate positive and negative control chicken sera were included in each plate. Fifty ARV negative serum samples (China Institute of Veterinary Drug Control) were tested at 450 nm to calculate the average (X) and Stdev (SD) of the OD450 nm values. The cut-off value was calculated according to the formula: cut-off value = X + 3SD. Forty-eight 1-week-old specific pathogenic free (SPF) chickens were divided into 3 groups. Fifteen chickens in group I were injected intramuscularly with inactivated vaccine of 104 TCID50/bird at 2 and 4 weeks old. In group II, 18 2-week-old chickens received ARV live virus at 104 TCID50/bird by eye drop. Fifteen chickens in group III were used as negative controls. Serum samples were collected at 7 weeks of age. Thirty-three sera from chickens infected or vaccinated with AVR were tested by NS-ELISA, P17-ELISA and NS-P17-ELISA separately. Using the whole virus antigen of ARV S1133, thirty-three sera from infected chickens and 33 from vaccinated chickens were tested by agar gel precipitation (AGP) as described (Ide and Dewitt, 1979). For specificity of ELISAs, serum samples from birds with NDV, AIV and IBV were tested by NS-ELISA, P17-ELISA and NS-P17ELISA separately. Statistical analyses of the data were performed by Kruskal–Wallis one-way analysis of variance with the Dunn’s post hoc with SigmaStat Version 3.0. The recombinant plasmids pGEX-4T-1-NS and pGEX-4T-1P17 were identified by PCR (data not shown). It was confirmed by DNA sequencing that the acquired recombinant plasmid contained complete NS and P17 genes, and the pGEX-4T-1-NS and pGEX-4T-1-P17 recombinant plasmids were successfully constructed. Analysis of the purified protein by SDS-PAGE revealed the expressed NS and P17 fusion proteins, with an approximate molecular mass of 66.2 kDa and 42.4 kDa, respectively. These were consistent with the expected size of the fusion proteins (Figs. 1A and B and 2), and the concentrations were 28 mg/mL and 46 mg/mL, respectively. Conditions for NS-ELISA, P17-ELISA and NS-P17-ELISA were standardized by using checkerboard titrations. The results show that, when the concentrations of NS, P17 and NS-P17 were 9.3 g/mL, 11.5 g/mL and 9.3 g/mL, respectively, at serum dilutions of 1:200, the NS-ELISA, P17-ELISA and NS-P17-ELISA gave a clear difference in absorbance values between positive and negative sera. According to 50 ARV negative serum samples, the cut-off value of NS-ELISA was 0.630, P17ELISA was 0.560 and NS-P17-ELISA was 0.640, respectively. NS-ELISA, P17-ELISA and NS-P17-ELISA differentiated ARV positive and negative antibodies in experimentally infected and vaccinated chickens (Tables 1 and 2). The NS-ELISA, P17-ELISA and NS-P17-ELISA were able to detect 88.9%, 61.1%, and 88.9%, respectively of ARV-infected serum samples, whereas the AGP test detected 55.6%. Detection rates on serum samples from the vaccinated SPF chickens were 6.7%, 0% and 6.7%, respectively for these nonstructural proteins. The AGP method detected 60.6% of samples positive for antibodies from ARV-vaccinated chickens. The NSELISA, P17-ELISA and NS-P17-ELISA showed no positive reactions with serum samples from birds with NDV, AIV and IBV, which indicated that these indirect ELISAs had good specificity for the detection of ARV antibody. The nonstructural NS and P17 proteins were expressed successfully and the purified proteins were used as ELISA antigens to detect antibodies against ARV. The purified antigens demonstrated good specificity in ELISAs. These antigens reacted specifically with
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Table 1 The OD450 values of individual serum samples. ELISA
NS
P17
NS-P17
NS
P17
NS-P17
NS
P17
NS-P17
Sera collected from chickens infected with ARV
0.781 1.279 0.744 1.086 0.606 0.711
0.686 0.518 0.871 0.795 0.462 0.669
0.808 1.057 0.771 1.068 0.629 0.779
1.167 0.822 0.945 0.674 0.752 0.732
0.947 0.689 1.013 0.483 0.688 0.512
1.076 0.842 1.075 0.726 0.843 0.820
1.395 0.574 1.099 0.899 0.720 1.198
1.049 0.483 1.158 0.427 0.548 0.909
1.118 0.612 1.216 0.917 0.683 1.105
Sera collected from chickens vaccinated with ARV inactive vaccine
0.431 0.617 0.294 0.379 0.470
0.382 0.418 0.521 0.373 0.358
0.298 0.437 0.518 0.272 0.365
0.428 0.223 0.697 0.582 0.285
0.307 0.311 0.491 0.468 0.344
0.353 0.341 0.693 0.265 0.525
0.306 0.346 0.317 0.407 0.516
0.490 0.252 0.385 0.508 0.386
0.326 0.389 0.354 0.476 0.622
Table 2 Detection rates positive for ARV in sera from chickens using ELISA and AGP. Detection method NS-ELISA P17-ELISA NS-P17-ELISA AGP
Infected with ARV *
88.9% 61.1% 88.9%* 55.6%
Vaccinated with ARV 6.7% 0% 6.7% 60.6%
* Detection with NS-ELISA and NS-P17-ELISA was significantly (P ≤ 0.05) different from P17-ELISA and AGP groups.
Fig. 1. Western blot analysis of ARV positive antiserum specific to the expressed NS (A) and P17 (B) fusion proteins. (A) Lane M, protein marker; lane 1, purified NS protein; lane 2, PGEX4T-1 induce at 4 h. (B) Lane M, protein marker; lane 1, PGEX4T-1 induce at 4 h; lane 2, purified P17 protein.
their respective serum samples, did not cross-react with any heterologous sera and exhibited negative reactivity with antisera against Newcastle disease virus, avian influenza virus and infectious bronchitis virus. Recombinant protein-based ELISA is less sensitive but more specific than the whole virus lysate-based ELISA. A previous report (Liu et al., 2002) demonstrated that the combined proteins-based ELISA, compared with the single protein-based
ELISA or whole virus lysate ELISA, could greatly reduce non-specific binding reactions and had a higher correlation with serum neutralization assays. The current results suggest that NS-ELISA, P17-ELISA and NS-P17-ELISA were able to detect antibody in serum samples from the ARV-infected chickens. Using NS and P17 proteins together as ELISA antigens did not increase the sensitivity of the assay. However, NS-ELISA, P17-ELISA and NS-P17-ELISA each was more sensitive than the AGP test (Table 2). The nonstructural proteins are expressed in large amounts in virus-infected cells, but they have not been detected in virions (Krug and Etkind, 1973). It has been known that ELISAs specific for nonstructural proteins of hepatitis C and equine influenza viruses might be able to distinguish vaccination from infection (Inoue et al., 1992; Birch-Machin et al., 1997; Ozaki et al., 2001). In our study, positive detection rates for NS-ELISA, P17-ELISA and NS-P17ELISA were high on the serum samples of infected chickens and vaccinated chickens (Table 2). Thus using NS and P17 as antigens in ELISA can differentiate between sera from vaccinated and infected chickens. The NS-ELISA, P17-ELISA and NS-P17-ELISA were found to be more sensitive than the AGP test (Table 2). This study showed that NS-ELISA was more sensitive than the P17ELISA. A plausible reason may be that the NS protein carries more broadly specific epitopes than the P17 protein. Further testing on a large numbers of serum samples from field cases will be required to confirm these findings. Serum samples from chickens infected with ARV were positive with ELISAs between 61.11% and 88.89%, however not all of the samples were positive. This may be because the antibody level was insufficient for detection by ELISA. The positive rate for 15 serum samples from vaccinated chickens was 0–6.7%. Not all the samples were negative. That may be because the vaccines were not purified, or because booster vaccinating makes more nonstructural proteins. Further studies will be carried out to elucidate these phenomena. In conclusion, the NS-ELISA and NS-P17-ELISA have advantages over the conventional ELISA, with less non-specific binding reactions and stable sensitivity. These could be used to distinguish vaccinated from infected chickens. Acknowledgements
Fig. 2. Purified avian reovirus nonstructural recombinant ARV proteins. Lane 1, P17; lane 2, NS; lane 3, protein marker.
The authors thank Dr. Herbert Van Kruriningen for critical reading of the manuscript. This study was supported by the National
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