Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Comprehensive Mapping of Common Immunodominant Epitopes in the West Nile Virus Nonstructural Protein 1 Recognized by Avian Antibody Responses

  • Encheng Sun,

    Affiliations The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China, Graduate School of Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China

  • Jing Zhao,

    Affiliations The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China, Graduate School of Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China

  • Nihong Liu,

    Affiliation The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China

  • Tao Yang,

    Affiliation The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China

  • Qingyuan Xu,

    Affiliation The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China

  • Yongli Qin,

    Affiliations The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China, Graduate School of Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China

  • Zhigao Bu,

    Affiliation The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China

  • Yinhui Yang,

    Affiliation Beijing Institute of Microbiology and Epidemiology, Beijing, People's Republic of China

  • Ross A. Lunt,

    Affiliation Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Australia

  • Linfa Wang,

    Affiliation Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Australia

  • Donglai Wu

    dlwu@hvri.ac.cn

    Affiliation The Key Laboratory of Veterinary Public Health, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China

Comprehensive Mapping of Common Immunodominant Epitopes in the West Nile Virus Nonstructural Protein 1 Recognized by Avian Antibody Responses

  • Encheng Sun, 
  • Jing Zhao, 
  • Nihong Liu, 
  • Tao Yang, 
  • Qingyuan Xu, 
  • Yongli Qin, 
  • Zhigao Bu, 
  • Yinhui Yang, 
  • Ross A. Lunt, 
  • Linfa Wang
PLOS
x

Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.

Introduction

West Nile virus (WNV) is a medically important pathogen that is prevalent in many areas around world, including Africa, Europe, Russia, the Middle East, India, Australia and North America [1]. It is serologically classified into the Japanese encephalitis virus (JEV) serocomplex, which includes WNV, JEV, Saint-Louis encephalitis virus (SLEV) and Murray Valley fever virus (MVEV) [2].

WNV is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. As such, the virus poses a risk to human health as well as the health of domestic animals and wildlife. In the first half of 2011, WNV was associated with 183 clinical neuroinvasive disease cases and 85 clinical non-neuroinvasive disease cases in humans [3]. The profile of WNV viremia following mosquito-borne infection of birds can vary greatly among different bird species [4][6]. Birds that sustained a viremic titer greater than 105.0 plaque forming units (PFU)/ml were considered infectious to C. pipiens and C. quinquefasciatus [7], [8]. Certain species of birds, including crows, geese, blue jays, ravens, chickens and house sparrows, are considered highly susceptible hosts for WNV, and may be involved in virus transmission through mosquito bites because they develop high levels of viremia after WNV infection [4][6], [9][21]. Deaths in American crows (AMCR) due to WNV infection were especially prevalent in New York City in 1999 [22], and crow mortality has been adopted as an epidemiological indicator to monitor WNV transmission in the United States and has proven useful in predicting an increased risk for human infection [23][25].

Fortunately, commercially raised chickens and turkeys have not been extensively affected by WNV, likely because they are predominantly raised indoors and have limited exposure to mosquito vectors [26]. Although chickens and turkeys can become infected with WNV when experimentally inoculated subcutaneously with WNV, infection results in low viral titers and does not cause clinical disease [27], [28]. Recently, mutations in the prM (prM-I141T) and envelope 40 (E-S156P) genes were shown to mediate the attenuated phenotype of the WNV TM171-03-pp1 virus variant in a chicken macrophage cell line [29]. Once mutated WNV strains were emergent, the strains with high virulence to chickens even causing fatality would also arise, so monitoring epidemiology of WNV infected chickens is necessary. Differences in the course of natural WNV infection in geese as compared to chickens have also been noted, as natural WNV infections could cause severe neurological signs and death in young domestic geese [6], [30]. While the role of domestic geese as a WNV reservoir in an outbreak in Israel is unknown, the infection rates of geese in the Sindbis District of the northern Nile Valley were similar to the rates in buffed-back herons (Bubulcus ibis ibis), doves (Streptopelia senegalensis senegalensis), and domesticated pigeons (Columbia livia) and twice the rate seen in domesticated chickens and ducks (Anas platyrhynchos), suggesting that geese may play a role in local WNV ecology [31].

Flavivirus nonstructural protein 1 (NS1) is an important nonstructural protein which plays critical roles in viral RNA replication and the development of flavivirus-associated diseases [32], [33]. Although NS1 protein is not present in the virion of flaviviruses, it can elicit non-neutralizing protective antibodies that inhibit infection through both Fc-γ receptor-dependent and -independent mechanisms [34]. Additional works has shown that passive administration of monoclonal antibody (mAb) against the NS1 protein or active immunization with the NS1 gene or protein confer protection from lethal flavivirus challenge [35][39], suggesting that immune responses targeted against the NS1 protein of flaviviruses play an important role in conferring immune protection. A single mAb against flavivirus NS1 protein also could effectively protect against lethal flavivirus challenge [34], [40][44]. While anti-E mAb-based therapies could be a promising strategy to control flavivirus infections, sub-neutralizing concentrations of anti-E antibodies have the potential to cause antibody-dependent enhancement (ADE) of flavivirus infections that complicate therapy [45][48]. Targeting the NS1 protein using protective antibodies may represent a promising alternative approach.

In future years, it is expected that migratory birds infected WNV will carry the virus to all parts of the United States as well as to Canada, the Caribbean, and Central and South America [49]. Mosquitoes capable of transmitting WNV to susceptible birds exist in each of these regions. The convergence of birds at scarce pools of water also facilitates virus transmission. Diagnostic platforms to monitor WNV infection in domestic avian species, including chickens, ducks and geese, which come into contact with wild waterfowl and birds would be extremely valuable. The rationale exploitation of the antigenic peptide epitopes in the WNV NS1 protein that are targeted by avian antibody responses could provide the basis for such a diagnostic platform.

The aim of our study was to identify immunodominant B-cell epitopes in the WNV NS1 protein that are targeted by the avian immune system using chicken, duck and goose antisera raised against recombinant WNV NS1 protein. The epitopes mapping described in this report will facilitate the development of diagnostic tests for the serological detection of WNV infection and the rationale design of subunit vaccines for WNV and other viruses of the JEV serocomplex.

Results

Titers of avian antisera

To generate NS1-reactive polyclonal antisera, chickens, ducks and geese were immunized three times with recombinant WNV NS1 protein. Antisera were collected two weeks after the third immunization for epitopes mapping experiments. Immediately prior to each immunization, serum was collected from each bird to measure the NS1-reactive antibody titers by immunofluorescence assay (IFA). As shown in Table 1, the WNV NS1-reactive antibody titers increased progressively with each sequential immunization in immunized chickens, ducks and geese. As expected, birds that were not immunized with NS1 protein did not have detectable levels of NS1-reactive antibodies at any time point (Table 1). Finally, the titers of the antisera from chicken, duck and goose determined by IFA were 1∶128, 1∶256 and 1∶512, respectively. Therefore, immunization with recombinant WNV NS1 protein elicits high-titer NS1-reactive antibodies in chickens, ducks and geese.

Comprehensive mapping of linear WNV NS1 B-cell epitopes by Western Blot (WB) using avian antisera

We next sought to identify linear epitopes within the WNV NS1 protein that are targeted by the avian antibody responses following immunization with recombinant WNV NS1 protein. We screened a series of 35 partially overlapping peptides covering the entire coding sequence of the WNV NS1 protein using the chicken, duck and goose antisera against purified WNV NS1 protein by WB. The NS1-derived peptides were expressed as MBP-fused polypeptides for screening. As shown in Table 2, the chicken antisera recognized twelve NS1 peptides, the duck antisera recognized nine NS1 peptides, and the goose antisera recognized six NS1 peptides in the series. Three peptides (NS1-3, 14 and 24) were recognized by antibodies in the sera of all three avian species following NS1 immunization; seven peptides (NS1-1, 3, 6, 9, 14, 20 and 24) were recognized by chicken and duck antisera; five peptides (NS1-3, 14, 18, 24 and 27) were recognized by duck and goose antisera; and four peptides (NS1-3, 14, 24 and 26) were recognized by chicken and goose antisera (Table 2). As expected, the control avian sera from unimmunized animals did not detectably react with any of the 35 MBP-fused polypeptides and MBP-tag only (data not shown).

thumbnail
Table 2. Identification of linear peptide epitopes in the WNV NS1 protein using antisera from different animals by WB.

https://doi.org/10.1371/journal.pone.0031434.t002

Confirmation of the identified WNV NS1 B-cell epitopes by ELISA using avian antisera

To confirm the reactivity of avian antibodies against the peptide epitopes identified by the WB, the candidate polypeptides were synthesized and screened by ELISA using the avian antisera. The chicken, duck and goose antisera displayed the same pattern of reactivity against the WNV NS1-derived polypeptides using an ELISA as was seen in the WB screening against the 35 MBP-fused polypeptides (Figure 1). Importantly, the antisera of all three avian species maintained reactivity against the three common NS1 peptide epitopes by ELISA (Figure 1, Peptide-NS1-3, 14 and 24), suggesting these linear epitopes may be of potential value for the development of serological diagnosis tests. As expected, the control avian sera from unimmunized animals did not detectably react with any polypeptide (data not shown).

thumbnail
Figure 1. Identification of NS1-derived linear peptide epitopes recognized by murine and avian antibodies generated following immunization with WNV NS1 protein.

Murine, chicken, duck and goose sera were collected following immunization with recombinant WNV NS1 protein and were evaluated for reactivity against a series of peptides derived from the WNV NS1 protein by ELISA. Each bar indicates antisera reactivity as determined by the mean absorbance at 492 nm and error bars indicate standard deviation.

https://doi.org/10.1371/journal.pone.0031434.g001

Evaluation of the conservation of three common epitopes among JEV serocomplex viruses

We next investigated whether the three linear epitopes in the WNV NS1 protein were specific to WNV or conserved among viruses of the JEV serocomplex. To this end, we aligned the NS1 amino acid sequences from viruses of the JEV serocomplex and antigenically related flaviviruses to identify the sequences corresponding to the three commonly recognized linear epitopes identified in the WNV NS1 protein (Figure 2, right panels). The corresponding polypeptides from other viruses of the JEV serocomplex were also synthesized and screened using the avian antisera raised against WNV NS1 protein to evaluate antibody cross-reactivity. The NS1-3 and 24 linear peptide epitopes, located at amino acids 21–36 and 231–246 of the WNV NS1 protein, were specific to WNV, as the three avian antisera did not react with polypeptides derived from the corresponding region of the JEV serocomplex members JEV, MVEV and SLEV (Figure 2A and C, left panels). The NS1-14 epitope, located at amino acids 131–146 of WNV, was common to all the JEV serocomplex viruses tested, as the avian antisera recognized the corresponding polypeptides derived from JEV, MVEV and SLEV (Figure 2B, left panel). As expected, the control avian sera from unimmunized animals did not detectably react with any polypeptide (data not shown).

thumbnail
Figure 2. Assessment of the specificity of common avian immunodominant epitopes by ELISA using polypeptides derived from homologous regions of viruses from JEV serocomplex viruses.

Chicken, duck and goose antisera immunized with WNV NS1 protein were tested for reactivity against three corresponding polypeptides from other JEV serocomplex viruses by ELISA to identify serotype- and group-specific B cell epitopes. For each polypeptide, the left panel displays the results of ELISA evaluating antibody binding to the JEV serocomplex peptides. Each bar indicates antisera reactivity as determined by the mean absorbance. The right panel depicts the sequence alignments used to identify corresponding polypeptides from representative strains of associated flavivirus isolates. The sequences from DENV1-4 and YFV are shown for comparison.

https://doi.org/10.1371/journal.pone.0031434.g002

Assessment of specificity of the three common peptide epitopes

Finally, we sought to determine whether antibodies generated against Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) could react with the three common WNV NS1 peptide epitopes that were targeted by avian species following immunization with WNV NS1 protein. AIV, NDV, DPV and GPV antisera did not react with the three WNV NS1 epitopes (NS1-3, 14 and 24), as indicated by an optical density (OD) lower than 0.2 at 492 nm (Figure 3). As expected, the control avian sera did not react with any polypeptide (data not shown). These data indicate that antibodies generated against other viruses, including AIV, NDV, DPV and GPV, do not cross react with the NS1-3, 14 and 24 linear WNV NS1 epitopes, and strongly suggest that these epitopes are specific to WNV and/or JEV serocomplex viruses.

thumbnail
Figure 3. Reactivity of AIV, NDV, DPV and GPV antisera against the common peptide epitopes recognized by avian antisera raised against the WNV NS1 protein.

The presence of antibodies reactive against the three common WNV NS1 epitopes in the indicated sera were evaluated by ELISA. Each bar indicates antisera reactivity as determined by the mean absorbance at 492 nm and error bars indicate standard deviation.

https://doi.org/10.1371/journal.pone.0031434.g003

Discussion

The differential diagnosis of WNV and related flaviviruses has long been a challenge. The practical application of serological diagnostic tests, including the virus neutralization test, the hemagglutination-inhibiting test, ELISA and IFA are limited by biosafety concerns [50]. Antibody cross-reactivity among members of the JEV serocomplex and other flaviviruses can confound the diagnostic outcome [51], [52], especially in geographic regions where several flaviviruses coexist [17]. Several studies have used a WNV NS1-specific mAb to detect natural infections among vaccinated populations and also to differentiate WNV from JEV infections in horses [53][56]. These studies illustrate that the NS1 protein can be exploited as a platform for the differential diagnosis of WNV and JEV serocomplex viruses. In addition to the potential utility of antibodies as diagnostic reagents, antibodies that bind epitopes within viral proteins can contribute to viral immunity in several ways, including virus neutralization and opsonization. The identification of antibody binding viral epitopes is required to understand antigenic composition of viral proteins and virus-antibody interactions at a molecular level.

In this study, we immunized three avian species using recombinant WNV NS1 protein and used the antisera to comprehensively map linear B-cell epitopes in the WNV NS1 protein. Following immunization, each avian species developed antibodies against some WNV NS1-derived polypeptides. We identified three WNV NS1-derived polypeptides that were commonly recognized by the antisera of all three avian species tested. We also identified other WNV NS1 peptide epitopes that were differentially recognized by chicken, duck and goose antisera following immunization. Some differences among the antibody specificities of mammalian and avian species were noted when we compared the NS1 epitopes recognized by avian antisera with epitopes previously identified using murine antisera and murine mAbs (Table 2) [57]. Despite some differences in the epitopes recognized among different species, we identified three epitopes that were commonly recognized by both mammalian (murine) and avian (chicken, duck and goose) antibodies. These common WNV NS1 epitopes correspond to WNV NS1 amino acids 21–36, 131–146 and 231–246, respectively, and are referred to as peptides NS1-3, 14 and 24. We confirmed that the NS1-3, 14 and 24 WNV NS1 peptides were commonly recognized by murine and avian antisera in an ELISA format. Further, the high OD at 492 nm that was achieved in the ELISA screening against the three common epitopes with each antiserum strongly suggests that these linear peptide epitopes are immunodominant.

Then we compared the WNV NS1 protein sequences with other viruses of JEV serocomplex and observed some amino acid residues differences between different strains of which some strains reacted with the antisera and others did not. This different reactivity between different polypeptides were associated with the low number of differentiating amino acids of the corresponding epitopes in NS1 protein as shown in Figure 2. The amino acids positions 35 (NS1-3), 237 and 240 (NS1-24) that may be crucial for reactivity of the two WNV-specific epitopes, because the antibodies generated following immunization with the WNV NS1 protein that recognized the NS1-3 and 24 epitopes did not cross react with the corresponding peptide sequences of other JEV serocomplex viruses (Figure 2A and C). More research is needed for further confirmation of these findings. Collectively, these results demonstrate that the two WNV-specific common immunodominant epitopes identified for the WNV NS1 protein can be used in tandem for the rationale design of serological tests to aid the differential diagnosis of WNV and other viruses of JEV serocomplex infection. Additionally, although NS1 protein is absent from the virion, it is expressed on the surface of infected cells and can elicit non-neutralizing antibodies during infection which are effective in protecting infected animals from lethal flavivirus challenge [29], [37]. Therefore, the mapping of antibody epitopes specific to WNV and common to viruses of the JEV serocomplex described in this work may be useful for the future development of avian common subunit vaccines.

Avian species, including crows, blue jays and ravens, are the main reservoir hosts in regions with endemic WNV. As such, these species typically are the initial source of WNV during epizootics occurring outside endemic areas [9], [13], [14], [20], [23][25]. Because of their close contact with migratory birds and waterfowl, ducks and geese may play an important role in the epidemiology of WNV. Recent works evaluating the connection between chickens and geese in WNV and infection with other flaviviruses suggested that chickens could serve as an appropriate sentinel animal for monitoring WNV epidemics [5], [6], [28], [30]. Additional, the three common epitopes identified did not react with any antiserum raised against AIV, NDV, DPV and GPV, confirmed the value of using them for serological diagnosis. As it is difficult to obtain sera from avian species that have been naturally infected with WNV or viruses of the JEV serocomplex in China, future works will be needed to confirm that the peptide epitopes identified using polyclonal sera generated against recombinant WNV NS1 protein also serve as antibody epitopes following bona fide infection with WNV.

In summary, our results identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the JEV serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. The data described in this work will guide the development and clinical application of serological diagnostic tests for WNV and other viruses of JEV serocomplex and also will contribute to avian subunit vaccines development.

Materials and Methods

Ethics statement

All animal studies were approved by the Review Board of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The Animal Ethics Committee approval number was Heilongjiang-SYXK 2006-032.

Avian species and proteins

WNV-negative avian species including chickens, ducks and geese were supplied by the Centre of Experimental Animals, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS). Purified WNV NS1 protein and 35 partially overlapping, MBP-fused polypeptides covering the entire WNV NS1 protein (NS1-1 to NS1-35) (Data S1) were generated in our laboratory as previously described [57].

Production and characterization of avian antisera

Chickens, ducks and geese (4 weeks old) were immunized subcutaneously and intramuscularly with purified recombinant NS1 protein in Freund's complete adjuvant (Sigma, St. Louis, MO, USA). The poultry received two boosters with purified NS1 protein in Freund's incomplete adjuvant at 2-week intervals. Animals were bled prior to each immunization, and one and two weeks after the final booster to determine the titer of NS1-reactive antibodies by IFA using WNV antigen slides. Serum collected two weeks after the final booster was used for the epitope mapping experiments described below. The IFA used to determine NS1-reactive antibody titers has been described previously [57][59]. Briefly, the primary antibodies were these sera from immunized and unimmunized chickens, ducks and geese. The IFA titers were determined with serial two-fold dilutions from 1∶2 to 1∶1024, and FITC-conjugated goat anti-chicken, rabbit anti-duck or rabbit anti-goose secondary antibodies were added at a 1∶100, 1∶50 and 1∶25 dilution, respectively.

Comprehensive mapping of epitopes using avian antisera by WB

A series of 35 partially overlapping peptides derived from the amino acid sequence of the WNV NS1 protein were expressed as MBP-fused polypeptides. Antibody reactivity against the MBP-fused polypeptides were screened by WB using chicken, duck and goose antisera. WB was performed essentially as described previously [57]. The primary antibodies from immunized animals or unimmunized animals were added at a 1∶100 dilution, and HRP-conjugated goat anti-chicken, rabbit anti-duck (LICOR Biosciences) or rabbit anti-goose secondary antibodies were added at a 1∶1,000, 1∶500 and 1∶200 dilution, respectively.

Synthesis of candidate epitopes for confirmatory screening of avian antisera by ELISA

The polypeptides which reacted with avian antisera by WB were synthesized for screening in an ELISA using the avian antisera (Table 3, Shanghai Bootech BioScience&Technology, China). The ELISA was performed as described previously, using synthesized polypeptides as coating antigen (100 ng/well) [57]. The irrelevant polypeptide (V5-Tag: GKPIPNPLLGLDST) and serum from unimmunized animals served as negative controls. All the sera were added at a 1∶100 dilution. HRP-conjugated goat anti-chicken, rabbit anti-duck (LICOR Biosciences) or rabbit anti-goose secondary antibodies were added at a 1∶2,000, 1∶1,000 and 1∶500 dilution, respectively. The cut-off value for the ELISA was determined as the mean OD492 nm values of negative control plus three standard deviations.

thumbnail
Table 3. Synthesized polypeptides used to identify linear peptide epitopes recognized by avian antisera.

https://doi.org/10.1371/journal.pone.0031434.t003

Analysis of antibody cross-reactivity against the common immunodominant epitopes using polypeptides from the NS1 protein of other JEV serocomplex viruses

Amino acid alignments were performed between the NS1 protein of WNV and other flaviviruses, including the JEV serocomplex members Kunjin virus, JEV, SLEV and MVEV, and the antigenically related flaviviruses DENV1–4 and YFV (Lasergene, DNASTAR Inc., Madison, WI). Based on these amino acid alignments, we identified polypeptide sequences in the JEV serocomplex viruses and related flavivirus that corresponded to the three common immunodominant epitopes identified within the NS1 protein of WNV. Polypeptides of these homologous NS1 regions from the related viruses were synthesized (Table 4, Shanghai Bootech BioScience&Technology, China). The synthesized polypeptides were evaluated for reactivity with avian antisera by ELISA as described above. An irrelevant polypeptide (V5-Tag) and sera from unimmunized avian species served as negative controls. The cut-off value for the ELISA was determined as the mean OD492 nm values of negative control plus three standard deviations.

thumbnail
Table 4. Synthesized polypeptides used to assess the cross-reactivity of antibodies that bind common avian immunodominant epitopes in the WNV NS1 protein with corresponding peptides derived from homologous regions of other JEV serocomplex viruses.

https://doi.org/10.1371/journal.pone.0031434.t004

The polypeptide sequences chosen were the predominant amino acid residues at each position according to the alignment results using all the available strains of certain virus in the NCBI Entrez protein database (http://www.ncbi.nlm.nih.gov/protein/).

Evaluation of specificity of the three common peptide epitopes

Six AIV, ten NDV, three DPV and three GPV antisera were tested for reactivity against the common epitopes by ELISA. The ELISA was performed as described above, using synthesized polypeptides (Peptide-NS1-3, 14 and 24) as coating antigens. An irrelevant polypeptide (V5-Tag) and sera from unimmunized avian species served as negative controls, the murine WNV NS1 protein positive serum served as positive control [57]. The cut-off value for the ELISA was determined as the mean OD492 nm values of negative control plus three standard deviations.

Supporting Information

Data S1.

The complementary oligonucleotide pairs encoding 35 overlapping, 16-mer peptides that encompassed the entire NS1 amino acid sequence from the WNV NY99 strain.

https://doi.org/10.1371/journal.pone.0031434.s001

(DOC)

Acknowledgments

The authors thank Dr. Peter Wilker for editing the manuscript.

Author Contributions

Conceived and designed the experiments: DLW. Performed the experiments: ECS JZ. Analyzed the data: TY QYX YLQ NHL. Wrote the paper: ECS JZ. Supplied the equine serum against WNV: RAL Supplied the results of IFA: YHY Supplied the WNV NS1 gene: ZGB Revised the manuscript: DLW LFW.

References

  1. 1. Garmendia AE, Van Kruiningen HJ, French RA (2001) The West Nile virus: its recent emergence in North America. Microbes Infect 3: 223–229.
  2. 2. Weissenbock H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder B, et al. (2002) Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis 8: 652–656.
  3. 3. Centers for Disease Control and Prevention (2011) West Nile Virus Human Infections in the United States.
  4. 4. Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, et al. (2003) Experimental Infection of North American Birds with the New York 1999 Strain of West Nile Virus. Emerg Infect Dis 9: 311–322.
  5. 5. Langevin SA, Bunning M, Davis B, Komar N (2001) Experimental infection of chickens as candidate sentinels for West Nile virus. Emerg Infect Dis 7: 726–729.
  6. 6. Swayne DE, Beck JR, Smith CS, Shieh WJ, Zaki SR (2001) Fatal encephalitis and myocarditis in young domestic geese (Anser anser domesticus) caused by West Nile virus. Emerg Infect Dis 7: 751–753.
  7. 7. Turell MJ, O'Guinn M, Oliver J (2000) Potential for New York mosquitoes to transmit West Nile virus. Am J Trop Med Hyg 62: 413–414.
  8. 8. Sardelis MR, Turell MJ, Dohm DJ, O'Guinn ML (2001) Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus. Emerg Infect Dis 7: 1018–1022.
  9. 9. Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, et al. (2001) Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States 1999. Emerg Infect Dis 7: 615–620.
  10. 10. Eidson M, Kramer L, Stone W, Hagiwara Y, Schmit K (2001) Dead bird surveillance as an early warning system for West Nile virus. Emerg Infect Dis 7: 631–635.
  11. 11. Komar N, Panella NA, Burns JE, Dusza SW, Mascarenhas TM, et al. (2001) Serologic evidence for West Nile virus infection in birds in the New York City vicinity during an outbreak in 1999. Emerg Infect Dis 7: 621–625.
  12. 12. Komar N, Burns J, Dean C, Panella NA, Dusza S, et al. (2001) Serologic evidence for West Nile virus infection in birds in Staten Island, New York, after an outbreak in 2000. Vector Borne Zoonotic Dis 1: 191–196.
  13. 13. Bernard KA, Maffei JG, Jones SA, Kauffman EB, Ebel G, et al. (2001) West Nile virus infection in birds and mosquitoes, New York State, 2000. Emerg Infect Dis 7: 679–685.
  14. 14. Work TH, Hurlbut HS, Taylor RM (1955) Indigenous wild birds of the Nile delta as potential West Nile virus circulating reservoirs. Am J Trop Med Hyg 4: 872–888.
  15. 15. Komar N, Lanciotti R, Bowen R, Langevin S, Bunning M (2002) Detection of West Nile virus in oral and cloacal swabs collected from bird carcasses. Emerg Infect Dis 8: 741–742.
  16. 16. Semenov BF, Chunikhin SP, Karmysheva V, Iakovleva NI (1973) Study of chronic forms of arbovirus infections in birds. I. Experiments with West Nile, Sindbis, Bhanja and Sicilian mosquito fever viruses [Russian]. Vestn Akad Med Nauk SSSR 28: 79–83.
  17. 17. Kuno G (2001) Persistence of arboviruses and antiviral antibodies in vertebrate hosts: its occurrence and impacts. Rev Med Virol 11: 165–190.
  18. 18. Lvov DK, Timopheeva AA, Smirnov VA, Gromashevsky VL, Sidorova GA, et al. (1975) Ecology of tick-borne viruses in colonies of birds in the USSR. Med Biol 53: 325–330.
  19. 19. Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, et al. (2000) Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol 37: 208–224.
  20. 20. Panella NA, Kerst AJ, Lanciotti RS, Bryant P, Wolf B, et al. (2001) Comparative West Nile virus detection in organs of naturally infected American Crows (Corvus brachyrhynchos). Emerg Infect Dis 7: 754–755.
  21. 21. Kramer LD, Bernard KA (2001) West Nile virus infection in birds and mammals. Ann N Y Acad Sci 951: 84–93.
  22. 22. Outbreak of West Nile-like viral encephalitis – New York, 1999. MMWR Morb Mortal Wkly Rep 48: 845–849.
  23. 23. Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, et al. (2001) Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States 1999. Emerg Infect Dis 7: 615–620.
  24. 24. Eidson M, Kramer L, Stone W, Hagiwara Y, Schmit K (2001) Dead bird surveillance as an early warning system for West Nile virus. Emerg Infect Dis 7: 631–635.
  25. 25. Julian KG, Eidson M, Kipp AM, Weiss E, Petersen LR, et al. (2002) Early season crow mortality as a sentinel for West Nile virus disease in humans, northeastern United States. Vector Borne Zoonotic Dis 2: 145–155.
  26. 26. Office International des Epizooties (2000) West Nile fever in the United States of America: in horses. Dis Information 13: 150–151.
  27. 27. Swayne DE, Beck JR, Zaki S (2000) Pathogenicity of West Nile virus for turkeys. Avian Dis 44: 932–937.
  28. 28. Senne DA, Pedersen JC, Hutto DL, Taylor WD, Schmitt BJ, et al. (2000) Pathogenicity of West Nile virus for chickens. Avian Dis 44: 642–649.
  29. 29. Langevin SA, Bowen RA, Ramey WN, Sanders TA, Maharaj PD, et al. (2011) Envelope and pre-membrane structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate. J Gen Virol.
  30. 30. Office International des Epizooties: West Nile fever in Israel in geese (1999) 12. 166 p. Disease Information.
  31. 31. Taylor RM, Work TH, Hurlbut HS, Rizk F (1956) A study of ecology of the West Nile virus in Egypt. Am J Trop Med Hyg 5: 579–620.
  32. 32. Lindenbach BD, Rice CM (1997) Trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71: 9608–9617.
  33. 33. Chung KM, Liszewski MK, Nybakken G, Davis AE, Townsend RR, et al. (2006) West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H. PNAS 103: 19111–19116.
  34. 34. Chung KM, Nybakken GE, Thompson BS, Engle MJ, Marri A, et al. (2006) Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and –independent mechanisms. J Virol 80: 1340–1351.
  35. 35. Lin YL, Chen LK, Liao CL, Yeh CT, Ma SH, et al. (1998) DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice. J Virol 72: 191–200.
  36. 36. Xu G, Xu X, Li Z, He Q, Wu B, et al. (2004) Construction of recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety and immunogenicity. Vaccine 22: 1846–1853.
  37. 37. Chung KM, Nybakken G, Thompson BS, Engle MJ, Marri A, et al. (2006) Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fc-γ Receptor-Dependent and-Independent Mechanisms. J Virol 80: 1340–1351.
  38. 38. Lin CW, Liu KT, Huang HD, Chen WJ (2008) Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus. Biotechnol Lett 30: 205–214.
  39. 39. Amorima JH, Porchiaa BF, Balan A, Cavalcantea RC, da Costac SM, et al. (2010) Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein. J Virol Methods 167: 186–192.
  40. 40. Henchal EA, Henchal LS, Schlesinger JJ (1988) Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. J Gen Virol 69: 2101–2107.
  41. 41. Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, et al. (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11: 522–530.
  42. 42. Schlesinger JJ, Brandriss MW, Walsh EE (1985) Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48. J Immunol 135: 2805–2809.
  43. 43. Schlesinger JJ, Foltzer M, Chapman S (1993) The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against YF encephalitis in mice. Virology 192: 132–141.
  44. 44. Lee TH, Song BH, Yun SI, Woo HR, Lee YM, et al. (2011) A Cross-Protective Monoclonal Antibody Recognizes a Novel 1 Epitope within the Flavivirus NS1 protein. J Gen Virol.
  45. 45. Peiris JS, Gordon S, Unkeless JC, Porterfield JS (1981) Monoclonal anti-Fc receptor IgG blocks antibody enhancement of viral replication in macrophages. Nature 289: 189–191.
  46. 46. Mehlhop E, Ansarah-Sobrinho C, Johnson S, Engle M, Fremont DH, et al. (2007) Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner. Cell Host Microbe 2: 417–426.
  47. 47. Cardosa MJ, Gordon S, Hirsch S, Springer TA, Porterfield JS (1986) Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement. J Virol 57: 952–959.
  48. 48. Pierson TC, Xu Q, Nelson S, Oliphant T, Nybakken GE, et al. (2007) The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe 1: 135–145.
  49. 49. Malkinson M, Banet C, Khinich Y, Samina I, Pokamunski S, et al. (2001) Use of live and inactivated vaccines in the control of West Nile fever in domestic geese. Ann N Y Acad Sci 951: 255–261.
  50. 50. Shi PY, Wong SJ (2003) Serologic diagnosis of West Nile virus infection. Expert Rev Mol Diagn 3: 733–741.
  51. 51. Koraka P, Zeller H, Niedrig M, Osterhaus AD, Groen J (2002) Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA. Microbes Infect 4: 1209–1215.
  52. 52. Williams DT, Daniels PW, Lunt RA, Wang LF, Newberry KM, et al. (2001) Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses. Am J Trop Med Hyg 65: 379–387.
  53. 53. Kitai Yoko, Shoda Mizue, Kondo Takashi, Konishi Eiji (2007) Epitope-Blocking Enzyme-Linked Immunosorbent Assay To Differentiate West Nile Virus from Japanese Encephalitis Virus Infections in Equine Sera. Clin Vaccine Immunol 14: 1024–1031.
  54. 54. Kitai Yoko, Kondo T, Konishi E (2010) Complement-dependent cytotoxicity assay for differentiating West Nile virus from Japanese encephalitis virus infections in horse sera. Clin Vaccine Immunol 17: 875–878.
  55. 55. Kitai Y, Kondo T, Konishia E (2011) Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: Effects of WN virus NS1 antibodies induced by inactivated WN vaccine. J Virol Methods 171: 123–128.
  56. 56. Kitai Y, Shirafuji H, Kanehira K, Kamio T, Kondo T, et al. (2011) Specific Antibody Responses to West Nile Virus Infections in Horses Preimmunized with Inactivated Japanese Encephalitis Vaccine: Evaluation of Blocking Enzyme-Linked Immunosorbent Assay and Complement-Dependent Cytotoxicity Assay. Vector-borne and Zoonotic Dis 11: 1093–1098.
  57. 57. Sun EC, Zhao J, Liu NH, Yang T, Ma JN, et al. (2011) Comprehensive mapping of WNV- and JEV serocomplex- specific linear B-cell epitopes from West Nile virus nonstructural protein 1. J Gen Virol.
  58. 58. Sun EC, Ma JN, Liu NH, Yang T, Zhao J, et al. (2011) Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage displayed random peptide library. BMC Microbiol 11: 160.
  59. 59. Sun EC, Zhao J, Yang T, Liu NH, Geng HW, et al. (2011) Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein. Virol J 8: 100.