International Immunopharmacology 19 (2014) 201–205

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Development of latex agglutination test with nucleoprotein as antigen for detection of antibodies to swine influenza virus☆ Rui-Hua Zhang a, Chun-Hong Li a, Wen-Xiao He b, Cun-Lian Wang a, Tong Xu a,⁎, Mei-Lin Jin c, Huan-Chun Chen c a b c

Key Laboratory of Preventive Veterinary Medicine, Department of Veterinary Medicine, Animal Science College, Hebei North University, Zhangjiakou 075131, PR China Artillery Training Base of General Staff, Zhangjiakou 075131, PR China Lab of Animal Infectious Diseases, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, PR China

a r t i c l e

i n f o

Article history: Received 15 December 2013 Received in revised form 11 January 2014 Accepted 28 January 2014 Available online 6 February 2014 Keywords: Latex agglutination test (LAT) H9N2 subtype Swine influenza virus Nucleoprotein

a b s t r a c t As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the generation of pandemic virus through reassortment. The broad susceptibility of pigs to influenza viruses emphasizes the importance of surveillance of swine influenza virus. Thus, A latex agglutination test (LAT) was developed for rapid detection of antibodies to swine influenza virus. The nucleoprotein (NP) gene of the H9N2 swine influenza virus isolated from local farms was cloned, and expressed in Escherichia coli. Reactivity of the expressed protein was confirmed by Western blot. Subsequently, the NP gene was purified and used as the diagnostic antigen to develop a NP-based LAT for detecting antibodies to swine influenza virus. The LAT is shown to be specific for swine influenza virus and does not cross-react with swine sera that have antibodies to other swine viruses. The NP-LAT and HI test had a high agreement ratio in detecting 10 serum samples from naïve pigs, 28 serum samples from experimentally infected and vaccinated pigs. Compared with the hemagglutination inhibition (HI) test, the corresponding specificity, sensitivity, and correlation were 92.9%, 94.1%, and 94.1%, respectively, in detecting 321 serum samples from vaccinated pigs. The NP-LAT developed in our laboratory is a rapid and simple test suitable for field monitoring of antibodies to swine influenza virus. We conclude that it was specific and sensitive and it has great application potential in China's long-term prevention and control of swine influenza virus. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Swine influenza is an acute respiratory disease caused by influenza A virus within the Orthomyxoviridae family. Infection of pigs with influenza A viruses is of substantial importance to the swine industry and to the epidemiology of human influenza [1]. The influenza A viruses are classified further into subtypes on the basis of the antigenicities of their surface glycoprotein hemagglutinin (HA) and neuraminidase (NA). To date, 16 HA (H1–16) and 9 NA (N1–9) subtypes of these viruses have been identified [2]. At present, the H9N2 subtype virus is a notable member of the influenza family because it can infect not only chickens, ducks and pigs, but also humans [3–9]. In China, the H9N2 virus was first isolated from a chicken in Guangdong province in 1992 and now is the most prevalent subtype of influenza virus in poultry in China [4]. Recent studies have demonstrated that H9N2 virus can infect pigs and cause significant morbidity and mortality [4,8]. In southern China, at least 2% of blood donors tested were positive for H9 antibodies ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Animal Science College, HeBei North University, Zhangjiakou 075131, PR China.Tel./fax: +86 313 4029336. E-mail address: [email protected] (T. Xu). 1567-5769/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2014.01.026

[3,10], suggesting that human infection with H9N2 occurs ubiquitously in this area. The frequent occurrence of influenza outbreaks among humans, swine, and birds has not only caused a huge economic loss but also posed a severe threat to human health. The broad susceptibility of pigs to influenza viruses emphasizes the importance of surveillance of swine influenza virus. Thus, a diagnostic assay is needed to efficiently diagnose swine influenza virus infection and at the same time, to detect antibodies induced by vaccines to validate vaccine efficacy. Currently, the serologic tests for swine influenza virus antibodies recommended by the World Organisation for Animal Health (OIE) are hemagglutination inhibition (HI) and an agar gel precipitation test (AGPT). This procedure, however, is labor intensive and time consuming. Moreover, hemagglutinin of swine influenza virus is subtype specific and also is undergoing constant mutation; HI assay is limited in its ability to detect all swine influenza virus subtypes. In serological monitoring, detection of antibodies against the nucleoprotein (NP) and the matrix protein (M1), which are highly conserved among influenza A viruses, should be attempted first, because this procedure can detect infections with various swine influenza virus subtypes. The AGPT is used widely because of its simplicity and broad specificity for the detection of influenza A viruses. However, with the AGPT, the final results are obtained after at least 2 days. Another serological test used commonly for influenza is the enzyme-linked immunosorbent assay (ELISA), which measures antibody responses to conserved proteins. ELISA results are obtained after

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several hours, and the ELISA procedure requires the use of special instruments and may be relatively labor intensive. In addition, ELISA may also show nonspecific reactions in some cases. Recently, a latex agglutination test (LAT) for detection of antibodies against the H5 subtype viruses was reported [11,12]. However, there are no reports on rapid and simple diagnostic methods for detecting antibodies against various subtypes of swine influenza virus. In this study, a NP based type-specific LAT was developed for detecting antibodies against all subtype influenza A viruses in our laboratory, and it was used in the diagnosis and serologic epidemiological investigations of swine influenza virus. The assay is sensitive, specific, and relatively inexpensive. It has great potential to be widely used by the pig industry of Asia.

were purified and sequenced on an ABI Prism 377XL DNA sequencer. The sequence homology was analyzed using the Basic Local Alignment Search Tool (BLAST) program of GenBank from the National Center of Biotechnology Information. 2.5. NP protein expression and purification A recombinant expression plasmid which is named pGEX-KG/NP was obtained by inserting NP gene into a prokaryotic expression plasmid pGEX-KG (TaKaRa). The pGEX-KG/NP expression vector was used to transform BL21(DE3) E. coli, and expression of the GST-NP protein was induced by adding isopropyl-b-D-thiogalactoside (IPTG). Proteins were purified on a GST-affinity column and then dialyzed in phosphate-buffered saline (PBS) for 3 days [14].

2. Materials and methods 2.1. Virus The A/swine/HeBei/012/2008/(H9N2) virus was isolated by our laboratory from local farms in Hebei province of China in 2008. Viruses were subtyped by standard hemagglutination-inhibition and neuraminidaseinhibition tests. The viruses were passaged in embryonated chicken eggs and the allantoic fluids were harvested and used as stock viruses for further analysis. 2.2. Experimental sera H1N1, H3N2, and H9N2 positive sera were obtained from the Harbin Veterinary Research Institute. Positive sera for porcine reproductive and respiratory syndrome virus (PRRSV) and classic swine fever virus (CSFV) were obtained from the Harbin Veterinary Research Institute, positive sera for porcine pseudorabies virus (PRV) were obtained from the Lab of Animal Infectious Diseases, Huazhong Agricultural University. 14 pigs were experimentally infected by the oronasal route with 106TCID50 of MDCK cell culture-grown H9N2 influenza viruses in 1 ml cell culture medium using a nebulizer device (Wolfe Tory Medical) as previously described [13]. The study was approved by the Animal Care and Use Committee of the Hebei North University (Zhangjiakou, Hebei). All animal procedures followed the ethics guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals (1985). Prior to infection animals were tested seronegative for influenza antibodies in a commercial blocking ELISA (ID.Vet). Blood samples used in further serological studies were obtained on day 28 post inoculation (p.i.). 10 sera samples from naïve pigs (unvaccinated and showed no clinical episodes of infection;), and 14 anti-H9N2-positive sera (HI titer N24) from experimentally vaccinated pigs were prepared by our laboratory. Blood samples used in further serological studies were obtained on the 28th day. 2.3. Field sera A total of 321 porcine field sera samples were collected from vaccinated pigs in several field farms in China. 2.4. NP gene cloning and nucleotide sequence analysis Viral RNA was extracted from allantoic fluid using the RNA-SOLV® reagent RNA isolation solvent (Omega Bio-tek, Lilburn, GA) according to the manufacturer's instructions. For the amplification of the NP gene, we designed a primer pair: forward primer, 5′-GCG GAT CCG CGT CTC AAG GCA CCA AAC-3′ (containing the BamH I cleavage site) and reverse primer, 5′-CGC AAG CTT ATA GTC ATA CTC CTC TGC-3′ (containing the Hind III cleavage site). Then the complete open reading frame of the NP gene was amplified using reverse transcriptasepolymerase chain reaction (RT-PCR). The amplified DNA fragments

2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) SDS-PAGE analysis was performed to confirm the expression of the target protein. To determine the antigenicity of the expressed NP protein, Western-blot analyses were performed. The primary antibody was serum obtained from a H9N2 swine influenza virus-immunized pig. Goat-anti-swine IgG(immunoglobulin G) conjugated with horseradish peroxidase (HRP) (Southern Biotech Associates Inc.) was used as the secondary antibody. The membrane was developed by incubation with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide until the color developed sufficiently. 2.7. LAT The optimal antigen concentration for sensitizing latex beads and the concentration of BSA in the blocking buffer were determined by the following processes. Latex beads (0.7 μm, Kexin Company, Shanghai, China) were washed three times with 0.1 M carbonate buffer (pH 9.6) and 0.02 M phosphate buffer (pH 4.5) respectively. The bead was then suspended in 2% ethyl-dimethyl-amino-propyl carbodiimide (EDAC) and incubated at room temperature for 4 h and centrifuged at 5000 ×g for 10 min. The supernatant was carefully aspirated out. After washing another three times with 0.01 M boric acid buffer (pH 8.4), the antigen which was serially diluted twofold with PBS (10 mmol L−1; pH 7.4) from 1:2 to 1:1024 was added and incubated for 4–5 h to sensitize latex beads by passive adsorption. To block nonspecific binding, the beads were blocked with 0.1 0.5, 1.0, 1.5% bovine serum albumin (BSA) for 30 min at 37 °C and centrifuged. Then blocked beads were suspended in a storage buffer [5% glycerol, and 0.1% sodium azide (NaN3) in phosphate buffered solution (PBS); pH 7.4] and used to react with either PBS (1:1) or anti-H9N2-positive serum to determine the optimal antigen concentration and the concentration of BSA in the blocking buffer (matrix). In detail, a 15-μL aliquot of sensitized beads was mixed with a 15-μL aliquot of PBS or anti-H9N2-positive serum, stirred gently, and observed for any agglutination reaction within 5 min. Test results were scored as rapid agglutination of 100% of sensitized latex beads with obvious ring formation (++++); agglutination of 75% of sensitized latex beads with some level of ring formation (+++); agglutination of N 50% of sensitized latex beads but with no ring formation (++); fine-particle agglutination, usually involving b25% of sensitized latex beads and interpretation questionable (+); and no visible agglutination greater than that of the negative control sample (−). Agglutination reactions + to ++++ were considered positive results. 2.8. Specificity and reproducibility of the LAT To determine the specificity of the LAT, the cross-reaction was evaluated with anti-H3N2-positive sera, anti-H1N1-positive sera, and anti-H9N2-positive sera and with serum samples positive for

R.-H. Zhang et al. / International Immunopharmacology 19 (2014) 201–205 Table 1 Homology analysis of the nucleotide sequences comparing the cloned NP gene and the NP gene from other representative influenza virus strains. Source

Locus

Homology ratio (%)

A/swine/Yangzhou/1/2008(H9N2) A/swine/Taizhou/5/2008(H9N2) A/chicken/Hebei/C4/2008(H9N2) A/environment/Hunan/5-38/2007(H9N2) A/Swine/HongKong/2429/98 (H3N2) A/duck/Ontario/05/00 (H3N2) A/TW/3446/02(H3N2) A/Swine/Hong Kong/2405/98 (H3N2) A/Shanghai/P1/2009(H1N1) (A/turkey/Kansas/4880/1980 (H1N1) A/swine/Northern Ireland/38(H1N1) A/swine/Cambridge/39(H1N1) A/chicken/Shantou/3900/2002(H5N1) (A/swine/Henan/wy/2004(H5N1)

HM998923 HM998915 GQ202048 GU474607 AF400757 GQ249058 DQ415331 AF400755 AB539740 EU742639 U04855 U04856 CY029175 DQ997255

98% 98% 99% 99% 81% 80% 82% 81% 83% 84% 84% 84% 97% 97%

PRRSV, PRV, and CSFV. For the reproducibility of the LAT, the latex beads sensitized with three batches of NP protein diluted to the same protein concentration were used to evaluate a total of 12 serum samples which cover a whole range of antibody levels, from negative to highly positive. 2.9. Comparison with the HI test Further evaluations of the specificity and sensitivity of the LAT in detecting swine influenza virus antibodies were carried out by testing 359 serum samples including 321 field serum samples from vaccinated pigs; 14 serum samples from experimentally infected pigs; 14 serum samples from experimentally vaccinated pigs; and 10 serum samples (0 titer by HI test) from naïve pigs. HI assays were performed as described [15]. 3. Results 3.1. Nucleotide sequence analysis The nucleotide sequence data from this study were deposited in the GenBank (the accession number is CY063663). The cloned nucleotide sequence of the NP gene was analyzed and compared with the NP gene sequences of representative swine influenza virus strains from

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GenBank (Table 1). The homology ratio is 80%–99% among influenza virus subtypes H1N1, H3N2, H5N1, and H9N2. 3.2. Protein expression, purification, and Western blot The NP gene was cloned into a prokaryotic vector pGEX-KG and expressed in E. coli system as GST-NP fusion protein with an apparent molecular mass of approximately 82 kD (56KD/NP + 26KD/GST) on SDS-PAGE (Fig. 1A). The expressed GST-NP protein was purified via GST-affinity column and approximately 56-kD purified NP protein was analyzed by SDS-PAGE (Fig. 1B). Then purified NP protein immunogenicty was confirmed by Western blot analysis using immune sera from H9N2 swine influenza virus vaccinated pig and a goat anti-pig secondary antibody. A specific 56 kDa band was detected in the purified NP protein, while there was no detectable band in the lysate of E. coli containing the pGEX-KG vector plasmid (Fig. 1C). 3.3. LAT The optimal concentrations of antigen and BSA in the blocking buffer to be used in NP-LAT were determined by matrix titration with antigen NP concentration ranging from 0.8 mg/ml (1:2 dilution) to 0.00156 mg/ml (1:1024 dilution) and BSA dilution ranging from 0.1% to 1.5%. The amount of NP protein for NP-LAT was determined to be 0.2 mg/ml (1:8 dilution) because it is the concentration with no autoagglutination and the highest titer for positive sera. A concentration of 1% BSA in the blocking buffer was determined as the best to discriminate positive from negative sera with minimized false-positive or falsenegative results. 3.4. Specificity and reproducibility of the LAT For the specificity test, the NP-LAT was used to detect a panel of sera positive for various viral diseases. The LAT showed positive result with sera positive for swine influenza virus subtypes H3N2, H1N1, and H9N2, and it did not react with sera positive for PRRSV, PRV, and CSFV (Table 2). The result indicating the NP-LAT was highly specific. To test reproducibility of the LAT, sera samples with antibody levels ranging from negative to highly positive were tested repeatedly with the latex beads sensitized with three batches of NP protein. Data

Fig. 1. (A) SDS-PAGE analysis of recombinant NP protein expressed in E. coli, stained with Coomassie brilliant blue R250. Lane M, protein molecular weight markers; Lane 1, lysate of E. coli with pGEX-KG/NP induced by IPTG; Lane 2, lysate of E. coli with pGEX-KG induced by IPTG. (B) SDS-PAGE analysis of purified NP protein, stained with Coomassie brilliant blue R250. Lane M, protein molecular weight markers; Lane 1, NP protein purified with a GST-column. (C) Western blotting detection of NP protein with swine sera positive for swine influenza virus H9N2. Lane M, protein molecular weight markers; Lane 1, lysate of E. coli containing the pGEX-KG vector plasmid. Lane 2, NP protein purified with a GST-column.

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Table 2 LAT reactions against different serum samples. Serum samples AntiH3N2

AntiH1N1

AntiH9N2

AntiPRRSV

AntiPRV

AntiCSFV

Negative

LAT +++

+++

++++









Note: PRRSV = porcine reproductive and respiratory syndrome virus, PRV = porcine pseudorabies virus, CSFV = classical swine fever virus, LAT = latex agglutination test; ++++ = very strong agglutination; +++ = strong agglutination; − = no visible agglutination.

showed there were no significant differences among the results for each sample. 3.5. Comparison with the HI test The agreement ratio was employed to compare differences between the NP-LAT and HI test in evaluating 28 sera samples from experimentally infected and vaccinated pigs. At 4 weeks after infection or vaccination, all 28 sera from both infected and vaccinated groups tested positive with HI test. By contrast, by NP-LAT, all 14 sera samples from the infected group tested positive, 13 sera samples from the vaccinated group were NP-antibody positive, and 1 sera sample from vaccinated group was NP-antibody negative. Sera from all 10 experimentally naïve pigs were negative in both NP-LAT and HI assays (Fig. 2). 3.6. Application of NP-LAT with field sera In testing 321 sera samples from vaccinated pigs in several field farms in China, 307 samples tested positive (inhibition titer, N24), and 14 samples tested negative (inhibition titer, b 24) by HI test. When tested by NP-LAT, 289 samples were positive, and 18 samples were negative from the 307 HI-positive samples, and 1 sample was positive and 13 were negative from the 14 HI-negative samples. Compared with that of HI, the specificity of the LAT was 92.9%, the sensitivity of the LAT was 94.1%. The correlation between the two tests was 94.1% (Table 3). 4. Discussion The NP of influenza virus is a type-specific antigen which is believed to be identical among the various strains of each type of influenza virus,

so the NP-based test can theoretically detect anti-influenza virus antibodies irrespective of the subtypes of the viruses. But the potential for influenza NP has been underappreciated because it is counterintuitive that an internal antigen would robustly induce an antibody. However, it is reported that both natural infection with influenza virus and vaccination with recombinant NP elicit NP-specific antibodies [16,17], so the detection of anti-NP antibody is applicable to serologic studies. In this study, the NP-based LAT was developed for detecting antibodies to all types of swine influenza viruses. For LAT, when sensitized by highly concentrated antigen, the latex beads can react with both PBS and standard positive antibodies. Normally, spontaneous agglutination with PBS and strong reactivity with standard anti-swine influenza virus-positive serum can be observed when high concentrations of antigen are used, so in this study, the amount of NP protein for NP-LAT was determined to be 0.2 mg/ml (1:8 dilution). Furthermore, the sensitivity of LAT was greatly influenced by the concentrations of BSA in the blocking buffer. Through repeated and systematic experimentation, the optimal parameters for the BSA in the blocking buffer were determined to be 1%. In our study, the NP-based LAT was highly specific and sensitive for detecting 28 serum samples of experimentally vaccinated or infected pigs (Fig. 2). The 28 serum samples are all positive by HI test (titer ≥ 24). With NP-LAT, all 14 samples from the infected pigs tested positive, and of the 14 vaccinated samples, 13 samples tested positive and the last, 1 sample, was negative. We demonstrated that the NP-LAT using E. coli expressed recombinant NP as the antigen and HI test had a high agreement ratio. For further evaluation of the specificity and sensitivity of the NP-LAT, 321 field serum samples from vaccinated pigs were detected by HI test and NP-LAT. The results showed that 307 samples tested positive (inhibition titer, N24), and 14 samples tested negative (inhibition titer, b 24) by HI test. When tested by NP-LAT, 289 samples were positive, and 18 samples were negative from the 307 HI-positive samples, and 1 sample was positive and 13 were negative from the 14 HI-negative samples (Table 3). It showed relatively higher agreement ratio in serum samples from experimentally infected and vaccinated pigs than in field serum samples from vaccinated pigs (Fig. 2 and Table 3), the reason is that maybe some of the negative field samples are likely to be true negative for infection or waning of antibody production. Comparison studies between the NP-based LAT and HI test for detecting the field serum samples from vaccinated pigs indicated that the specificity of the LAT was 92.9%, the sensitivity of the LAT was 94.1%, and the two tests had a high agreement ratio.

Fig. 2. Comparison of HI and NP-LAT in detecting experimental sera. (A) HI antibody titers in pig sera, positive sera by HI: titer ≥ 24; (B) Detection of antibodies to the NP by LAT in pig sera, positive sera by NP-LAT: (1) 4 on the ordinate axis = very strong agglutination; (2) 3 on the ordinate axis = strong agglutination; (3) 2 on the ordinate axis = moderate agglutination; (4) 1 on the ordinate axis = agglutination; negative sera by NP-LAT: 0 on the ordinate axis = no visible agglutination.

R.-H. Zhang et al. / International Immunopharmacology 19 (2014) 201–205 Table 3 Correlation between the LAT and HI tests in detecting 321 field serum samples from vaccinated pigs. Result

HI positive HI negative LAT total (%)

No. of field samples

HI total (%)

NP-LAT positive

NP-LAT negative

289 1 290

18 13 31

307 14 321

Note: specificity of LAT (13 of 14; 92.9%); sensitivity of LAT (289 of 307; 94.1%); correlation ([289 + 13] / 321; 94.1%). LAT = latex agglutination test; HI = hemagglutination inhibition.

This LAT is simple, rapid, and easy to perform, with no requirement for special equipment or skilled personnel, and results can be obtained within several minutes. Furthermore, the LAT would be much less expensive than other methods. Overall, the results of our study show the generation of a simple, cost-effective, sensitive, and specific LAT for serologic assays, which have a great potential for use in field practice monitoring for antibodies against swine influenza virus. This method could also be employed for epidemiologic surveillance. Acknowledgments This work was supported by the Natural Science Foundation of Hebei Province, China (Grant no.: C2011405002), the Natural Science Research Key Programs of Educational Department of Hebei Province (Grant no.: ZD20131045) and Key Programs of Hebei North University. References [1] Landolt GA, Karasin AI, Phillips L, Olsen CW. Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs. J Clin Microbiol 2003;141:1936–41.

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Development of latex agglutination test with nucleoprotein as antigen for detection of antibodies to swine influenza virus.

As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the generat...
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