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The NS2 protein of Bluetongue virus (BTV) is an important non-structural protein and plays important roles in viral replication and assembly. In this study, one monoclonal antibody (mAb), 4D4, was raised against BTV8 NS2. Phage display technology was used and identified the consensus binding motif SNYD recognized by mAb 4D4. To define the minimal region required for antibody binding, a panel of synthetic peptides encompassing SNYD derived from the BTV8 NS2 was then used to more specifically define the 4D4 epitope as 149RSNYDV154. Furthermore, amino acid sequence alignments of different BTV serotypes and other orbiviruses suggested that this epitope is highly conserved among the BTV serotypes. The mAb reagent generated in this study may be applied to the development of BTV diagnosis and surveillance programs and the epitope defined here can lead to important insights into how BTV might interact with the sheep's immune system.

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.

Coxsackievirus B3 (CVB3) was thought to be the most common causative agent of life-threatening viral myocarditis. Coxsackievirus B3 strain CC (CVB3-CC) was isolated in China; however, no sequence data are available. The 1A and 3D regions of CVB3-CC were sequenced and phylogenetic analysis was done with reference to ten other CVB3 strains and all 36 prototype strains of human enterovirus B (HEV-B). Sequence analysis showed that the 1A gene region of CVB3-CC consisted of 207 nucleotides, encoding 69 amino acids; and the 3D gene region was comprised of 1386 nucleotides, encoding 462 amino acids. Variation analysis showed that the 3D gene of CVB3 strain CC varied the least among the two regions. Phylogenetic tree analysis of the 1A and 3D regions indicated that CVB3-CC clustered together with CVB3 Nancy strain suggesting that there may be a close evolutionary relationship between the two strains. Incongruity was observed between the non-structural protein gene and the structural protein gene trees, according ...

The sigmaB/sigmaC-encoding genes of muscovy duck reovirus (DRV) S12 strain were cloned, sequenced, and expressed in Escherichia coli. The sigmaC-encoding gene of DRV showed only 21-22% identity to that of avian reovirus (ARV) at both nucleotide and amino acid level. The sigmaB-encoding gene of DRV comprised 1163bp with one open reading frame (ORF). The ORF comprised 1104bp and encoded 367 amino acids with a predicted molecular mass of 40.44 kDa. A zinc-binding motif and a basic amino acid motif were found within the predicted amino acid sequence of sigmaB. The identities between the S12 and ARV were 59.3-64.0% and 60.9-62.5%, respectively, at the nucleotide and deduced amino acid levels. Phylogenetic analysis of the sigmaB-encoding gene sequence indicated that S12 separated as a distinct virus relative to other avian strains. The expressed sigmaB/sigmaC fusion proteins in E. coli could be detected, approximately 45 and 50kDa, respectively, by duck anti-reovirus polyclonal serum. In addition, an ELISA (sigmaB-sigmaC-ELISA) using the expressed sigmaB-sigmaC proteins as coating antigen for detection of antibodies to DRV in ducks was developed. In comparison with the virus neutralization test and agar gel immuno-diffusion test (AGID), the sigmaB-sigmaC-ELISA showed perfect specificity and sensitivity. The sigmaB-sigmaC-ELISA did not react with the antisera to other duck pathogens, implying that these two proteins were specific in recognition of DRV antibodies. Taken together, the results demonstrated that sigmaB-sigmaC-ELISA was a sensitive and accurate method for detecting antibodies to DRV.

Severe acute respiratory syndrome (SARS) was caused by a novel virus now known as SARS coronavirus (SARS-CoV). The discovery of SARS-CoV-like viruses in masked palm civets ( Paguma larvata ) raises the possibility that civets play a role in SARS-CoV transmission. To test the susceptibility of civets to experimental infection by different SARS-CoV isolates, 10 civets were inoculated with two human isolates of SARS-CoV, BJ01 (with a 29-nucleotide deletion) and GZ01 (without the 29-nucleotide deletion). All inoculated animals displayed clinical symptoms, such as fever, lethargy, and loss of aggressiveness, and the infection was confirmed by virus isolation, detection of viral genomic RNA, and serum-neutralizing antibodies. Our data show that civets were equally susceptible to SARS-CoV isolates GZ01 and BJ01.

VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7. Western blot analysis showed the same result. Indirect immunofluorescence results indicated that two of the MAbs present different response spectrums with BTV1~24 serotypes. These results indicate that these MAbs may be good candidates for a specific diagnostic method and functional exploration of the VP7 protein.

Bluetongue virus (BTV) VP5 protein is an important antigenic protein which is centrally involved in serotype determination and the virus entry process. Very little is known about the B-cell epitopes on the BTV VP5 protein recognized by humoral immune responses. In this study, we generated five BTV16 VP5 protein-specific monoclonal antibodies (MAbs), named 3B11, 2B10, 1H7, 4A6 and 3G9, and defined the linear epitopes recognized by MAbs using a series of peptides expressed as maltose-binding protein (MBP)-fusion polypeptides. Three novel linear B-cell epitopes were identified: 3B11 and 3G9 recognized the motif ITANTREIQHIKEE; 2B10 recognized the motif LSGID; and 4A6 recognized the motif STMVKEYRQKIDALKA. Exact sequences corresponding to the three motifs identified were found in the BTV16 VP5 protein ((310)ITANTREIQHIKEE(323), (265)LSGID(269) and (188)STMVKEYRQKIDALKA(203)). These motifs represent the minimal linear peptide sequence required for MAb reactivity, as binding of each MAb was abolished when additional amino acids were removed from the amino and carboxy termini of the peptide. Amino acid sequence alignment indicated that three epitopes were totally conserved among different BTV16 strains. The MAbs generated along with identified epitopes will be useful for examining VP5 protein function and the development of epitope-based marker vaccines against BTV.

The present study identified a linear B-cell epitope in the Eastern equine encephalitis virus (EEEV) E2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an EEEV E2 specific monoclonal antibody (mAb) 7C11 and defined L/F-E/R-Y-T-W-G/R-N-H/W-P as the consensus binding motif. A sequence (321EGLEYTWGNHPP332) encompassing this consensus motif was found in the EEEV E2 glycoprotein and synthesized for further epitope confirmation. Meanwhile, the corresponding epitope peptides in E2 protein of associated alphaviruses were synthesized for specificity identification. Results showed the mAb 7C11 and murine antisera all reacted strongly against the synthesized polypeptide of EEEV antigen complex, but no reaction with Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) was detected. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against EEEV.

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.

We report here the complete genomic sequence of the Chinese bluetongue virus serotype 1 (BTV1) strain SZ97/1. This work is the first to document the complete genomic sequence of a BTV1 strain from China and represents the second complete sequence of BTV1 in the world. The sequence information provided here will help determine the geographic origin of Chinese BTV1 and provide data to facilitate future analyses of the genetic diversity and phylogenetic relationships of BTV strains.

We report here the complete genomic sequence of the Chinese bluetongue virus serotype 16 (BTV16) strain BN96/16. This work is the first to document the complete genomic sequence (segments 1 to 10) of a BTV16 strain. The sequence information provided herein will help determine the geographic origin of BTV16 and define the phylogenetic relationship of BTV16 to other BTV strains.

West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes in WNV NS1. We screened eight NS1-specific mAbs and antisera (polyclonal antibodies; pAbs) from mice immunized with recombinant NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. The screen using mAbs identified four WNV-specific (including Kunjin virus) epitopes, located at aa 21–36, 101–116, 191–206 and 261–276 in WNV NS1. However, using pAbs, only three WNV-specific epitopes were identified, located at positions 101–116, 191–206 and 231–246. Two of these epitopes (aa 21–36 and 261–276) had different reactivity with mAbs and pAbs. The knowledge and reagents generated in this study have potential applications in differential diagnostics and epitope-based marker vaccine development for WNV and viruses of the JEV serocomplex.

Background The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.

Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2K d -restricted CTL epitope, and the latter is an H-2D d -restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.

In this study, pCAGG-ChIL2 plasmid DNA containing the chicken interleukin-2 (ChIL-2) gene was used to prepare DNA-chitosan nanoparticles (CNPs). The CNPs prepared were spherical, with mean diameters between 100 and 200 nm, have a positive surface charge, and could protect DNA against DNase I degradation. The CNPs prepared were successfully used to transfect the Df-1 cell line with almost no cytotoxicity. CNPs prepared at an amino group to phosphate group ratio (N/P ratio) of 16 provided the highest transfection efficiency (1.1%) in medium with a pH of 6.5. When pCAGG-ChIL2 CNPs were administered to chickens simultaneously with a DNA vaccine against Newcastle disease virus (NDV), haemagglutination inhibition antibody titers and serum interferon-γ (IFN-γ) levels were significantly higher than in chickens immunised with the NDV DNA vaccine alone (p

IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-gamma gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of BALB/c mice with rEMIL-18, nine monoclonal antibodies (mAbs) against equine interleukin-18 (EIL-18) were obtained and characterized. These mAbs recognized different epitopes on equine mature interleukin-18 (EMIL-18) protein based on their reactivity with two peptides containing different amino acid sequences and one of these mAbs has neutralization activity against EIL-18 in an IFN-gamma-induction assay.

This report documents the first sequence analysis of the entire M1, M2, and M3 genome segments of the muscovy duck reovirus (DRV) S14. The complete sequence of each of the three M gene segments was determined. The M1 genome segment was 2283 nucleotides in length and was predicted to encode muA protein of 732 residues. The Escherichia coli expressed M1 transcripts generated a 108kDa protein, as expected for muA. A cleavage product of muA, muA1, could be detected by Western blotting with duck anti-reovirus and mouse anti-muA polyclonal serum. muA was distributed diffusely in the cytoplasma and nucleus of transfected Vero cells, which provides evidence that muA might be functional related to the mammalian reovirus (MRV) mu2. The M2 gene was 2155 nucleotides in length and was predicted to encode muB major outer capsid protein of 676 amino acids. The M3 genome segment was 1996 nucleotides in length and was predicted to encode a muNS protein of 635 amino acids. It was unexpectedly found that 5'-termini of the M1 and M2 genes ended with 5'-ACUUUU and 5'-UCUUUU, respectively, instead of 5'-GCUUUU, which is present on most mRNAs of other avian reoviruses (ARV). The UCAUC 3'-terminal sequences of the S14 M1, M2, and M3 genome segments are shared by DRV, ARV, and MRV. Alignment of the DRV muA-, muB-, and muNS-encoding genes with ARV revealed 72.9-73.9%, 67.1-69.6%, and 69.4-70.8% nucleotide identity, respectively. The amino acid sequence homology between DRV and ARV ranged from 85.3 to 86.2% (muA), 75.0 to 76.5% (muB), and 78.4 to 79.8% (muNS). Phylogenetic analyses of the M1, M2, M3, and S-class [Kuntz-Simon, G., Le Gall-Recule, G., de Boisseson, C., Jestin, V., 2002. Muscovy duck reovirus sigmaC protein is a typically encoded by the smallest genome segment. J. Gen. Virol. 83, 1189-1200; Zhang, Y., Liu, M., Hu, Q.L., Ouyang, S.D., Tong, G.Z., 2006a. Characterization of the sigmaC-encoding gene from muscovy duck reovirus. Virus Genes 36, 169-174; Zhang, Y., Liu, M., Ouyan, S.D., Hu, Q.L., Guo, D.C., Han, Z., 2006b. Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses aggregated the same specified genogroup in Orthoreovirus Genus II. Arch. Virol. 151, 1525-1538] genome segments suggests that DRV and ARV share a recent common ancestor and that the two lineages have subsequently undergone host dependent evolution.

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.

Background The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. Results In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. Conclusion The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.