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
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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 . 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 . 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 . 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
R.-H. Zhang et al. / International Immunopharmacology 19 (2014) 201–205
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 .
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 . 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
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 . 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
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.
R.-H. Zhang et al. / International Immunopharmacology 19 (2014) 201–205
Table 2 LAT reactions against different serum samples. Serum samples AntiH3N2
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 (%)
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  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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