Abstract
Figures and Tables
Fig. 2
Effect of bursal peptides (BPs) on antigen-specific immune responses. Mouse sera were collected on days 0, 7, 14, and 21 after the first immunization. Serum hemagglutination inhibition (HI) antibody titers (A) were analyzed by HI assay at 0, 7, 14, and 21 days; antigen-specific antihemagglutinin (anti-HA) (B), antibody subtypes IgG1 (C), and IgG2a (D) titers were analyzed by enzyme-linked immunosorbent assay on days 7 and 21. Data are presented as mean ± SD values from five replicates. AIV, avian influenza virus; PBS, phosphate-buffered saline; HA, hemagglutinin; OD, optical density. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine alone.
![jvs-19-817-g002](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-g002.jpg)
Fig. 3
Effect of bursal peptides (BPs) on cell-mediated immune responses in mice. Mouse sera (n = 5) were collected on days 7 and 21 post-immunization; Th1 (interferon gamma)-type cytokines (A) and Th2 (interleukin 4)-type cytokines (B) levels were measured by using commercial enzyme-linked immunosorbent assay kits. (C) Mouse splenic lymphocytes were isolated from spleen (n = 5) on day 21 post-immunization for cytotoxic T-lymphocyte (CTL) assay. (D) Flow cytometry analysis of the total CD3+T cells and subsets CD4+ and CD8+T cells from the splenic lymphocytes of immunized mice. Data are presented as mean ± SD values from five replicates. PBS, phosphate-buffered saline; AIV, avian influenza virus; IFN-γ, interferon gamma; IL-4, interleukin-4. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine.
![jvs-19-817-g003](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-g003.jpg)
Fig. 4
Detection of avian influenza virus (AIV) titers in lungs of H9N2 AIV-challenged mice by quantitative polymerase chain reaction (PCR) and TCID50 assay (TCID50, 50% tissue culture infective dose). Lung samples from individual mice in each group (n = 5) were collected on days 3, 5, and 7 post-challenge with 2.5 × 106 TCID50 AIV A/Chicken/Jiangsu/JS-1/2002 (H9N2). Lung virus copies were determined by quantitative PCR. Each lung sample was diluted to 1 mL with phosphate-buffered saline (PBS), panels A–C in Fig. 4 shows the viral genomes (copies) presented per milliliter of total RNA in lung tissue of individual mice in each group (n = 5). Panels D–F in Fig. 4 shows the viral titers as plaque-forming units (PFU) per milliliter as determined by TCID50 assay. Data are presented as mean ± SD values from five replicates. BP, bursal peptide. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine alone.
![jvs-19-817-g004](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-g004.jpg)
Fig. 5
Pathology changes in lung sections harvested at days 3, 5, and 7 after challenge and stained with H&E. Lung sections were obtained from mice (n = 5) in each group, fixed in buffered formaldehyde solution, and stained with H&E to assess inflammation and tissue damage after challenge. The arrows indicate representative pathological changes of the lung tissues: inflammation, perivascular and interstitial infiltrates. PBS, phosphate-buffered saline; BP, bursal peptide. 20×.
![jvs-19-817-g005](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-g005.jpg)
Fig. 6
Pathological scores of lung sections harvested at days 3, 5, and 7 after challenge. Histological scoring for virus-infected mice; mean values for each group are shown (n = 5). Histopathological changes were scored by an investigator “blind” to sample identity Scores were assigned as follows: a score of 1 indicates no pathology, 2 indicates perivascular infiltrates, 3 indicates perivascular and interstitial infiltrates affecting 20% of the lung lobe section, 4 indicates perivascular and interstitial infiltrates affecting 20% to 50% of the lung lobe section, and 5 indicates perivascular and interstitial infiltrates affecting 50% of the lung lobe section. PBS, phosphate-buffered saline; AIV, avian influenza virus; BP, bursal peptide.
![jvs-19-817-g006](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-g006.jpg)
Table 1
Animal groups and the experimental design
![jvs-19-817-i001](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-i001.jpg)
PBS, phosphate-buffered saline; TCID50, 50% tissue culture infective dose; AIV, avian influenza virus; BP, bursal peptide. *H9N2 AIV vaccine, commercial H9N2 avian influenza virus vaccine prepared with oil/water as an adjuvant. †A group of mice that was not immunized and not challenged was used as a blank control.
Table 2
Titers of plaque reducing neutralizing antibody in groups of mice
![jvs-19-817-i002](/upload/SynapseData/ArticleImage/0118jvs/jvs-19-817-i002.jpg)
Mouse sera on day 7 (first immunization) and day 21 (second immunization) were collected and analyzed. The 50% plaque reduction neutralizing titer (PRNT50) was the geometrical reciprocal of the sera dilution resulting in a 50% reduction; avian influenza virus (AIV)-neutralizing antibodies were detectable in 1:2 diluted mouse sera in the phosphate-buffered saline (PBS) group. Standard group is H9N2 inactivated vaccine (n = 5). Data are presented as mean ± SE. BP, bursal peptide. *p < 0.05 and **p < 0.01 compared to mice immunized with H9N2 inactivated vaccine.
Acknowledgments
References
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