Immunofluorescence Analysis of Duck plague virus gE protein on DPV-infected ducks

Background In previous studies, the expression and localization characteristics of duck plague virus (DPV) gE protein have been described in cultured cells, but the properties of DPV gE protein have not been reported in vivo. Immunofluorescence analysis had been used for the detection of virus antigen, but there was no report on the use of this technique for the detection of DPV gE. In this study, we investigated the distribution of DPV gE protein on DPV-infected ducks using polyclonal antibody raised against the recombinant His-gE fusion protein by indirect immunofluorescence assay (IFA). Results The recombinant gE protein was highly immunogenicity by ELISA, and the gE was used as an antigen for the preparation of polyclonal antibody, which could be used the first antibody for further experiment to study the distribution of DPV gE protein in DPV-infected tissues by indirect immunofluorescence assay. DPV gE protein were distributed in the immune organs (thymus, bursa of fabricius (BF), Harders glands, spleen), the digestive organs (liver, duodenum, jejunum, ileum), and the other parenchymatous organs (kidney, myocardium, cerebrum, and lung) of DPV-infected ducks, but the positive immunofluorescence signal was not seen in the muscle and pancreas. The lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes served as the principal site for the localization of DPV gE antigen. Moreover, the intensity of fluorescence increased sharply from 12 to 216 h post-infection (p.i.). Conclusions In this work, the immunogenicity of the recombinant gE protein was analyzed by ELISA, and we presented the distribution properties of DPV gE antigen in infected ducks for the first time, which may be useful for understanding the pathogenesis of DPV. These properties of the gE protein provided the prerequisite for further functional analysis.


Background
Duck plague (DP) is an acute contagious disease that is highly lethal in all ages of birds from the order Anseriforms (ducks, geese, and swans) [1]. The characterization of duck plague is tissue hemorrhage, digestive mucosal eruptions lesions of lymphoid organs and degenerative changes in parenchymatous organs [2]. Duck plague was difficult to monitor and control, because duck plague virus established an asymptomatic carrier state in both domestic and wild waterfowls that was detectable only during the intermittent shedding period of the virus [3]. Duck plague has resulted in significant economic losses in commercial duck industry due to high mortality rate and decreased duck egg production [4].
Glycoprotein E (gE) encoded by US8 from Alphaherpesvirinae had demonstrable effects on virulence and spread in the nervous system, and played important roles in determining the extents of cell-to-cell spread, perhaps by binding a ligand while on the surface of an infected cell and signaling through its cytoplasmic sequences to affect gene expression in the infected cells [5,6]. The duck plague virus (DPV) gE protein is a 490amino acid glycoprotein protein encoded by US8 gene. At present, some studies showed immunofluorescence assay (IFA) method had been widely used for the detection of specific pathogen, virus, and bacteria [7,8], but no report was available on the use of this technique for the detection of duck plague virus (DPV) gE protein. In this study, using polyclonal antibody raised against the recombinant His-gE fusion protein, the distribution of DPV gE was investigated in paraffin-embedded tissues of experimentally DPV-infected ducks by indirect immunofluorescent staining method.

Results
Expression and Immunogenicity of DPV gE protein DPV gE protein was overexpressed in E.coil Rosetta, and purified as an antigen for antibody development (Figure 1.A). The result of ELISA indicated that the recombinant protein was observed to be highly immunogenic. On 7 days, the OD450 nm value obtained was 0.702, while unimmunized ducks showed the OD450 nm value of 0.247, and the OD 450 nm value of immunized ducklings with DPV commercial attenuated vaccine strain was 0.681. At 28 days, the OD450 nm values reached maximum value (2.009) after inoculation, and the antibody titers of DPV gE protein continued to have a high level for 126 days ( Figure 2).

The purification of the His-gE antiserum and Optimum conditions of IFA
The rabbit polyclonal antiserum raised against the recombinant gE protein were prepared, and the His-gE antiserum was purified, the IgG was collected (Figure 1.B), and examined by SDS-PAGE (Figure 1. A). The purified gE antiserum was subsequently used as primary antibody in indirect immunofluorescent staining method. The optimum conditions of IFA for DPV gE antigen detection were as follows: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide (H 2 O 2 ) in methanol for 45 min, antigen recovery was performed on microwave with citrate buffer solution (0.01 M, PH 6.0) for 20 min, the sections were incubated with 10% normal goat serum for 2 h at 37°C, and incubated the primary antibody (1:100) for 60 min at 37°C. The secondary antibody was diluted 1:150, and incubated for 45 min at 37°C.

Detection of DPV gE antigen
Following experimental DPV infection, the tissues were obtained from the infection group and the controls. The systemic distribution of DPV gE antigen by IFA was summarized in Table 1

Specific detection
Specific fluorescent signal for DPV gE antigen was intensely found in immunological organs and digestive organs from the DPV-infected ducks, while no positive signal was located in the tissues of mock-infected ducks, and Duck hepatitis virus (DHV), Marek's disease virus (MDV), New type gosling viral enteritis virus (NGVEV)infected birds during the whole experiment (Table 2).
The glycoprotein gE play an important role in determining the virulence of duck plague virus. In our study, DPV gE fusion protein, with a relative molecular mass of 74 kDa, was expressed in E. coli Rosetta. The recombinant protein was surveyed to be highly immunogenic by ELISA, and purified as an antigen for antibody preparation. Previous study indicated that the gE envelope glycoprotein is very significant immunodominant antigen for the development of new antibodies, and the rabbit anti-His-gE polyclonal antibody had high reactivity and specificity for DPV gE [9,10]. It demonstrated that the antiserum could be used as the primary antibody for detecting the distribution of gE protein on DPV-infected ducks. IFA was a highly specific and sensitive diagnostic method for detecting infectious diseases [11][12][13]. In this report, the DPV gE antigen on DPV-infected ducks was examined by IFA. The result demonstrated that DPV gE antigen was extensively present in immunological organs and digestive organs. DPV gE antigen was detected firstly in Lymphoid organs (Bursa of Fabricius, thymus, spleen, and Harderian gland) (Figure 3.). The thymus and BF were the primary immune organs, where the differentiation and maturation of T lymphocytes and B lymphocytes generated, respectively [14,15]. Harderian gland and spleen were the secondary immune organs. There are many interstitial aggregations of B-dependent lymphoid cells in the Harderian gland [16][17][18], and spleen is an abundant source of immunocompetent cells (T and B lymphocytes) [19][20][21]. T and B lymphocytes played important roles in mediating immune responses. It could offer that the lymphoid organs were damaged seriously, after immune responses to heteroantigen in lymphoid organs of ducks infected with duck plague virus.
The immunofluorescence signal appeared in the liver and small intestine (including the duodenum, jejunum, and ileum) as early as 8 h p.i. (Figure 4.). These results indicated that the DPV-infected ducks occurred mucosal damage of digestive organs after the normal defence mechanism was damaged. These results of the present study were in accordance with previous studies. Shen had reported that the bursa of Fabricius, thymus, spleen, liver, and intestine were the main target organs of DPV in acute DPV cases [22]. Furthermore, the intensity of positive immunofluorescence signal of DPV gE antigen in various tissues increased sharply from 12 to 216 h p.i. Duck plague virus proliferated quickly in lymphoid organs and the digestive organs, finally, the other parenchymatous organs (kidney, myocardium, lung and cerebrum) were invaded by blood circulation (Figure 4.). In addition, DPV gE could induce cell fusion and promote cell-to-cell spread of the virus in infection process [23,24] and the release of the virus [25,26], so that it was easy to accelerate the progress of the infection. And gE could promote the virus to transport the central nervous system in infection [27], which was damaged. These data also indicated that DPV would infect a variety of tissues in ducks, and additional replication in these sites caused a major viraemia.

Conclusions
In conclusion, we described for the first time the basic characteristics of DPV gE distribution in the tissues of experimentally DPV-infected ducks by indirect immunofluorescence assay. From these results of immunofluorescence studies, it concluded that DPV gE mainly located in the immunological organs and digestive organs. This research will provide new insights into understanding the pathogenesis of DPV. Further studies will be aimed at constructing of DPV gE mutant to study the function of DPV gE.

Materials and methods
Virus strain, Immunogenicity Analysis of gE Protein, and Antibody preparation DPV CHv strain was a high-virulence field strain, obtained from Avian Disease Research Centre of Sichuan Agricultural University. DPV commercial attenuated vaccine strain was provided by Key Laboratory of Animal Disease and Human Health of Sichuan Province. The gE fusion protein was expressed in Escherichia coli as our laboratory described previously [10,28,29]. His-gE recombinant protein was expressed in Escherichia coli Rosetta induced by isopropy1-β-Dthiogalactopyranoside (IPTG) at 30°C for 4.5 h. Over expressed His-gE fusion protein were purified using a Ni-NTA affinity chromatography.
And the immunogenicity analysis of His-gE protein was detected by ELISA as our laboratory described previously [30]. The procedures were as follows: 96-well plates were coated with the purified gE protein (1:100) overnight at 4°C and blocked with 1% BSA in PBST (PBS containing 0.05% Tween-20) for 1 h at 37°C. After three washings with PBST, the sera from immunizing ducklings (at 3, 7, 28, 35, 42, 49, 56, 70, 126 days after immunizing the gE protein) were diluted (1:160) to the wells and incubated for 1 h at 37°C. Finally, after washing three times, plates were incubated with HRP-conjugated goat anti-duck IgG (1:1000) (KPL, USA) for 1 h at 37°C. Then, the antibody was removed, and the plates were washed 3 times with PBST. The reaction was developed with the 3'3'5'5-tetramethylbenzidine (TMB) substrate with H 2 O 2 . The optical density was measured at 450 nm using Bio-Rad 860 reader (Bio-Rad, USA) after stopping  the reaction with 2 mol/L H 2 SO 4 . The control groups were immunized with DPV commercial attenuated vaccine strain as positive group and phosphate-buffered saline solution (PBS) as negative group. Then, the rabbit polyclonal antiserum raised against the recombinant gE protein were prepared as described previously [10]. And the antiserum was purified by ammonium sulfate precipitation and High-Q anion-exchange chromatography using BioLogic LP system [31]. At first, the column was washed in 100% solvent A (HPLC water) for 30 min. The column was equilibrated in 100% solvent A (20 mM Tris-HCl) for 20 min, then the sample (the antiserum) was loaded in the columm, and the columm was equilibrated in 100% solvent A (20 mM Tris-HCl) for 10 min. Finally, the column was eluted with linear gradient of 100%A (20 mM Tris-HCl)/0%B(1 M NaCl) to 0%A (20 mM Tris-HCl)/100%B(1 M NaCl) for 30 min and the sample was collected and analyzed by SDS-PAGE.

Experimental animals and sampling
Thirty five 30-day old Cherry Valley ducks (not vaccinated against DPV) were divided randomly into two groups. Twenty-five ducks were intramuscularly inoculated with DPV CHv strain at a dose of 0.2 ml containing 10 3 MLD (minimum lethal dose). The remaining ducks were intramuscularly with 0.2 ml PBS (0.01 M, pH 7.4) instead of virus and used as the negative control. The ducks were housed in isolation units in a biosecure building and provided with a commercial duck diet ad libitum. At 2, 4, 8, 12, 24, 72 h postinfection (p.i.), and later every 1 day p.i. until they died (216 h p.i.), two ducks were randomly taken from the infection group, and one duck was randomly selected in the controls and euthanatized at each time point. These tissues (Harderian gland, Bursa of Fabricus (BF), thymus, duodenum, jejunum, ileum, liver, spleen, pancreas, myocardium, lung, kidney, cerebrum, and muscle) were collected from DPV-infected ducks and fixed by 4% paraformaldehyde in 0.1 mol of phosphate buffer (PH 7.4). In addition, these tissue sections of duck hepatitis virus (DHV), Marek's disease virus (MDV), and new type gosling viral enteritis virus (NGVEV)-infected birds were provided by Key Laboratory of Animal Disease and Human Health of Sichuan Province.
Optimization and application of Immunofluorescence assay (IFA) Tissues were fixed, and processed for paraffin embedding, and cut at 4 um thickness. The sections were dewaxed in xylene and re-hydrated in PBS (0.01 M, PH 7.4) for 5 min. Endogenous peroxidase activity was blocked by immersing the slides in 0.3% hydrogen peroxide (H 2 O 2 ) in methanol for 45 min (A) or 3% H 2 O 2 in methanol for 15 min(B). Then the sections were submitted to antigen retrieval in represents microwave antigen retrieval in citrate buffer solution (CBS, 0.01 M, pH 6.0) for 20 min(A), 0.1% trypsase for 10 minutes at 37°C(B), or no recovery. After washing three times with (PBS containing 0.05% Tween-20, 0.01 M, PH 7.4), the slides were incubated with 10% normal goat serum or 10% bovine serum albumin for 2 h at 37°C. The blocking serum was tapped off, and the sections were covered with the purified rabbit-gE polyclonal antibody without dilution, at dilutions of 1:50, 1:100, or 1:200 in PBST containing 1%BSA, and incubated in a humidified chamber for 1 h at 37°C. After washing with PBST three times, the slides were covered with the fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG diluted 1:50, 1:100, 1:150, or 1:200 in PBST containing 1%BSA for 45 min, 60 min, or 90 min at 37°C. Finally, the sections were lightly counterstained with 0.01% Evans blue at room temperature, and examined through a fluorescent microscope (Nikon 80i).

Specificity assessment
The tissues of PBS-infected ducks, and other virus (DHV, MDV, NGVEV) infected birds were detected by IFA, respectively.